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UDC: 612.647:611.018.1:611.61
Quantitative analysis of the nephron during
human fetal kidney development
Marija Dakovi-Bjelakovi*, Slobodan Vlajkovi*, Rade ukuranovi*,
Svetlana Anti*, Goran Bjelakovi†, Dejan Miti‡
University of Niš, Medical Faculty, *Institute of Anatomy, †Department of Internal
Medicine, Clinical Centre, ‡Clinic of Gynecology and Obstetrics, Niš
Background. The development of human kidney is a complex process. The number, shape,
size, and distribution of nephrons as functional units in a kidney, provide some important
information about the organization of the kidney. The aim of this study was to extend the
knowledge of the developing human kidney by studying nephrons in the kidney's cortex
during gestation. Methods. Kidney tissue specimens of 32 human fetuses, the gestational
age from IV lunar month (LM IV) to LM X, were analysed. Specimens were divided in ten
groups based on gestational age. Stereological methods were used at the light microscopic
level to estimate the volume densities of the corpuscular and tubular components of the
nephron in the cortex of the developing human kidney. Results. Nephron polymorphism
was the main characteristic of the human fetal kidney during development. In younger fetuses, just below the renal capsule, there was a wide nephrogenic zone. It contained the
condensed mesenchyme and terminal ends of the ureteric bud. Nephrons, in the different
stages of development, were located around the ureteric bud which branched in the cortical nephrogenic zone and induced nephrogenesis. More mature nephrons were located in
the deeper part of the cortex, close to the juxta-medullary junction. During gestation, nephrogenesis continually advanced, and the number of nephrons increased. Glomeruli
changed their size and shape, while the tubules changed their length and convolution. Renal cortex became wider and contained the more mature glomeruli and the more convoluted tubules. The volume density of the tubular component of the nephron increased continually from 10.53% (LM IVa) to 27.7% (LM X). Renal corpuscles changed their volume
density irregularly during gestation, increasing from 13% (LM IVa) to 15.5% (LM IVb).
During the increase of gestational age, the volume density of corpuscular component of the
nephron decreased to 11.7% (LM VIII), then went on increasing until the end of the intrauterine development (LM X) when corpuscles occupied 16.73% of the cortical volume.
The volume density of the developing nephrons (corpuscular and tubular portion) showed
the significant positive correlation (r = 0.85; p<0.01) with gestational age. Conclusion.
The present study was one of few quantitative studies of the human developing nephron.
Knowledge about the normal development of the human kidney should be important for the
future medical practice.
Key words:
embryo and fetal development; kidney; nephrons.
Introduction
Organogenesis of the kidney begins with the successive appearance of pronephros, mesonephros, and metanephros (1). The development of the metanephros, or permanent kidney, is a complex and gradual process. Metanephros, the definitive human kidney, is the result of the reciprocal inductive interactions between the two primordial
mesodermal derivates: ureteric bud, which is an epithelial
outgrowth of the Wolffian duct, and metanephric blastema,
which is a group of mesenchymal cells (2, 3). The nephron
arises from the metanephric mesoderm, and the collecting
ducts, calyces and renal pelvis arise from the ureteric bud
that branches off the mesonephric duct (1). Metanephros
first becomes apparent at about the 5th week of intrauterine
life. From the 8th to the 36th weeks of gestational age, nephrogenesis is continually induced by the ureteric bud ramifications (4, 5). The nephron progresses through four devel-
Dakovi-Bjelakovi M, et al. Vojnosanit Pregl 2005; 62(4): 281286.
282
opmental stages, which are described as vesicle, commashaped (or lipped) and S-shaped stages, developing capillary loop, and finally, the maturing glomerulus stage (1, 6).
As the ureteric bud branches in the metanephric mesenchyme, it induces the mesenchymal cells so as to condense
around the tip of a bud forming a cap-like structure. Shortly
after the aggregation, each mesenchymal condensate undergoes the phenotypic change into a hollow sphere or the vesicle
of the epithelial cells. Following these phenotypic changes, the
epithelial cells at the lower pole of the vesicle, opposite to the
ureteric bud, change their shape, and then a cleft appears,
forming a comma-shaped body. Next, the second cleft is
formed at the opposite pole of the comma-shaped body, which
is known as an S-shaped body. Microvessels invade the lower
cleft. The cells surrounding these vessels are the source of the
glomerular vasculature (6, 7). At the onset of the comma
shaped stage, and continuing into the S-shaped stage, the morphological segmentation of the developing nephron into the
glomerular and tubular regions can be recognised. The Sshaped body begins to elongate and joins the ureteric bud (8,
9). The tubules go on elongating, thus becoming more convoluted. The glomerular cells keep on differentiating until they
acquire their adult features forming the maturing stage glomeruli in developing kidneys. The number, shape, size, and the
distribution of nephrons provide an important information
about the organization of the kidney (10). Fetal glomerular
features change during the development (11).
There are many reasons for the importance of studying
the kidney development. The human developing kidney,
however, has not been quantified yet. It has been proposed
that many renal disease stages in the adults should be determined by the events that occur during the fetal development (12, 13). The fetal metanephric kidney produces dilute
urine, which is a major component of the amniotic fluid,
which makes an essential aqueous environment for the
symmetrical growth of a fetus, and the correct lung development. Any factor that obstructs the urine production by
the kidneys could result in fetal abnormalities (14).
The aim of this study was to extend the knowledge of
the human developing kidney by quantifying the changes in
fetal kidney cortex during nephrogenesis and the maturation
of the nephron, and by studying the relative growth of the
nephron components during gestation.
4
suggest the congenital malformations. Specimens were divided in ten groups based on gestational age. This kind of division allows one group to be the control - one to another. The
human fetal kidney tissue fragments were fixed in 10%
phosphate-buffered formaldehyde, treated with the conventional techniques, and embedded in paraffin. The sections
about 5 Pm thick each were stained with haematoxylin–eosin
(HE). Inside the referent space, which was represented by the
renal cortex, corpuscular and tubular components of the nephron were quantified. Sampled sections were analysed using
the light microscope with M42 multipurpose test-system.
The volume density (Vvf) was determined by analysing 2025 microscopic fields per kidney. The number of
points hitting the developing corpuscles (vesicles, commashaped and S-shaped form, C-shaped form, fully developed
corpuscles) and tubules were counted. Volume density was
calculated by the formula Vv=¦Pf/ ¦Pt, where Vv is the
volume density, Pf is the test-point number hitting the component of the nephron, and Pt is the test-point hitting the tissue. Means, standard deviations and 95% confidence intervals (CI) of the renal corpuscles and tubules for the consecutive gestational ages were calculated. Comparisons
between groups were analysed using Student’s t-test. The
relationships between the volume density of nephron and
gestational age were estimated using Pearson’s correlation
coefficient. All data were analyzed using SPSS 10.0.
Results
The main characteristic of the human fetal kidney
during development was the nephron polymorphism. Nephrons were of different size and shape. In the younger fetuses, just below the renal capsule, there was a wide nephrogenous zone. It contained the metanephric mesenchyme
and terminal ends of ureteric bud. The nephrons with the
mature glomeruli and the proximal and distal tubules, were
located at the juxta-medullary junction (Figure 1).
cm
ub
Methods
Thirty two human fetal kidneys of single pregnancies
were studied. The fetuses were obtained from spontaneous
abortions caused by prematurity or perinatal asphyxia, from
the Institute of Pathology, Clinical Center Niš. The last menstrual period and crown-rump length of the fetuses were used
to determine the gestational age which ranged from IV lunar
months (LM IV) to LM X. In order to record a generation of
new nephrons correctly, we divided IV, V and VI lunar
months into the first and second half (a and b) (15). There
were no apparent abnormalities of the fetuses, which could
Fig. 1 Light micrography of human fetal kidney (13th
week of gestation).
The nephrogenic zone next to the kidney surface contains condensed mesenchyme (cm), terminal ends of the ureteric bud (ub)
and the earliest stages of nephron development. More mature
nephrons are located next to developing medulla. HE, u 100
4
Mesenchymal cells condensed around the ampullar
extremity of ureteric bud. Nephrons in the different stages
of development were located around the ureteric bud which
branched in the cortical nephrogenic zone and induced
nephrogenesis (Figures 23).
V
cm
283
During gestation, nephrogenesis continually advanced,
and the number of nephrons increased. Glomeruli changed
their size and shape, as well as the length and the convolution of tubules. The nephrogenic zone became thin and the
immature structures could be seen below the capsule. The
cortex became wider and contained the more mature glomeruli and progressively the more convoluted tubules (Figure 4).
C
S
V
Fig. 2 Ureteric bud branching induces nephrogenesis.
Metanephric mesenchyme condenses (cm) around the tip of the
ureteric bud. Different stages of nephron differentiation are located around the developing collecting tubule: vesicle (V), Sshaped body (S) and mature glomerulus (G). HE, u 400
G
G
Fig. 3 Comma-shaped body is located close to the
kidney surface.
Erythrocytes are placed within the lower cleft (arrows). Nephrons
with mature glomerulus (G) and proximal and distal tubules are located
in the inner cortical region. HE, u 400
Fig. 4 Light micrography of part of the cortex in human
fetal kidney (23rd week of gestation). HE, u 100
At the end of nephrogenesis, the nephrogenic zone
disappeared completely. The renal cortex of the newborn
contained the mature nephrons which were smaller than
those in the adult one.
The estimations of volume density (expressed in percentages) of the corpuscular and tubular components of
nephrons are shown in Table 1. The volume density of a tubular component increased from 10.53% (LM IVa) to
27.7% (LM X). The increase was statistically significant at
LM Va, LM Vb and LM X (p<0.05; p<0.001, respectively).
The volume density of renal corpuscles increased significantly at LM IVa (p<0.05). It rised from 13% (LM IVa) to
15.5% (LM IVb). The volume density of the corpuscular
components of the nephron decreased at the some time with
the statistically significant increase of the volume density of
tubules at LM V.
Table 1
Volume density of the corpuscular and tubular components of the nephron during gestation
Corpuscles Vv (%)
Lunar Gestational age Sex
month (weeks)
Mean r SD
(95%CI)
IVa
12.114
1F/2M
13.00 r 0.89
10.7915.21
IVb
14.116
1F/2M
15.50 r 0.82*
13.4717.53
Va
16.118
1F/2M
14.03 r 0.42
12.9915.07
Vb
18.120
2F/1M
13.60 r 0.95
11.2315.97
VIa
20.122
1F/3M
13.06 r 1.22
10.0316.10
VIb
22.124
2F/2M
12.60 r 1.74
8.2716.93
VII
24.128
3F
12.33 r 0.49
11.1113.56
VIII
28.132
1F/2M
11.70 r 0.62
10.1513.25
IX
32.136
2F/1M
12.13 r 0.21
11.6212.65
14.1519.32
X
36.140
2F/1M
16.73 r 1.04†
F - female; M - male; Vv - volume density; CI - confidence interval.
*p<0.05; †p<0.01; ‡p<0.001
Tubules Vv (%)
Mean r SD
(95% CI)
10.53 r 1.50
6.8014.26
11.90 r 0.17
11.4712.33
13.80 r 0.92* 11.5216.08
16.03 r 0.21* 15.5216.55
19.37 r 1.10
16.6322.10
20.26 r 1.07
17.6122.92
20.37 r 0.99
17.9222.82
20.47 r 1.85
15.8725.62
21.13 r 0.57
19.7222.55
27.70 r 1.14‡
24.8830.52
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Its value went on decreasing until LM VIII (11.70%),
and then started to increase until the end of the intrauterine
development, at LM IX but not significantly, and at LM X
significantly (p<0.01), when the corpuscles occupied
16.73% of the cortical volume. The volume density of the
developing nephrons (corpuscular and tubular portion) increased from 23.53% at LM VIa to 44.43% at LM X (Figure 5) and showed the significant positive correlation (r =
0.85; p<0.01) with gestational age (Figure 6).
Vv (%) nephrons
50
40
30
Vv (%) tubules
20
Vv (%) corpuscules
10
0
14 16 18 20 22 24 28 32 36 40
Gestational age (w eeks)
Fig. 5 Volume density (%) of nephrons during gestation
y = 0.5465x +
r 2 = 0.7212
Vv (%) nephrons
50
40
30
20
10
0
12
16
20
24
28
32
36
40
Gestational age (weeks)
Fig. 6 Correlation between volume density of nephrons
and gestational age
Discussion
The knowledge about human fetal kidney development is still limited. Several studies emphasized the relation of the fetal kidney development, especially nephrogenesis, and the adult renal diseases. It was suggested that
kidney disease should be determined by the events that
occur during the fetal development (16, 17). Brenner et al.
(18) suggested that congenital nephron deficits predispose
individuals to hypertension later within the life. It was
also shown that any disturbance of the ureteric bud outgrowth during renal organogenesis and/or its branching
pattern might lead to renal malformation and various degrees of oligonephronia (19). This study was an effort to
contribute to the knowledge about the human developing
kidney, especially by studying the relative growth of
4
nephron components in the cortex of the human fetal kidney during gestation. It illustrated the sequential view of
the human glomerulogenesis in light microscopy. Hricak
et al. (20) determined how many glomeruli were present at
the different stages of human development. They found
that in the neonatal kidney the glomeruli occupied 18% of
the cortical volume, compared to 16.73% in our study.
Almeida et al. (21), in the quantitative study of glomeruli
at the second and third human gestational trimesters,
found the volume density increased from 18% to 20.40%.
Bertram et al. (9) analyzed the absolute and relative volumes of the developing rat kidney. They found that the
proportion of nephron tubules increased from 0 to 27%, as
compared to 27.70% in our study. By embryonic day 21
in the rat, which corresponded to the 40th week of gestation, the developing nephrons comprised 43.10% of the
metanephros compared to 44.43% in our study (9).
Osathanondh and Potter (22) divided the development
of a nephron into three periods. The first began with the
separation of the cells from the metanephric blastema that
would form a sphere, an ovoid vesicle and S-shaped tubule.
It ended with the establishing of the communication between the upper limb of the tubule and the collecting tubule
ampulla. The second period was characterized by the differentiation of the tubular part of the nephron in segments. The
third period went on until the loop gained its full length and
the convoluted tubules their maximal tortuosity. The first
two periods lasted about two weeks each, and the second
one extended up to the adult life.
Hinchliffe et al. (15) also proposed three periods in
human renal development. The first one was the population
period (1525 weeks) with the generation of new nephrons
as a dominant feature. The second one was the lag period
(2536 weeks) in which the immature nephron formation
was balanced by the increasing growth of the cortical component of the nephron. The third one was the growth period
(36 to 40 weeks), when the cortical nephron segment
growth became dominant in the induction of nephrons.
Population and lag periods coincided with the Potter’s second and third period.
The nephron number increased from 15 weeks gestation to 40 weeks. Hinchliffe et al. (15) found that the rate of
increase of nephron number was the greatest from weeks 15
to 17 and that it corresponded to the start of Osathanondh
and Poter’s second period (22). Our results were almost the
same. We found the statistically significant increase in the
volume density of renal corpuscles at LM IVb. During the
fetal development, from LM V to LM IX, which corresponded to Hinchliffe’s population and lag period, the significant increase of the volume density of tubules was accompanied with a slight decrease in the volume density of
renal corpuscles, but in accordance with the growth of the
cortical component of the nephron. At LM X, which corresponded to the Hinchliffe growth period, the volume density
of renal corpuscles and tubules increased statistically significantly too.
4
Conclusion
The present study was one of few quantitative studies of the human developing nephron. The knowledge
285
about the age-related normal developing kidney morphogenesis should be important for the future medical practice.
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4
Apstrakt
Dakovi-Bjelakovi M, Vlajkovi S, ukuranovi R, Anti S, Bjelakovi G, Miti
D. Vojnosanit Pregl 2005; 62(4): 281286.
KVANTITATIVNA ANALIZA NEFRONA TOKOM RAZVOJA BUBREGA
LJUDSKOG FETUSA
Uvod. Razvoj humanog bubrega kod oveka je složen proces. Broj, oblik, veliina i
raspored nefrona, kao funkcionalnih jedinica bubrega, pružaju znaajnu informaciju
o organizaciji bubrega. Cilj ovog istraživanja bio je proširenje saznanja o razvoju
bubrega kod humanog fetusa prouavanjem nefrona u bubrežnom korteksu tokom
gestacije. Metode. Analizovani su uzorci tkiva bubrega 32 humana fetusa gestacijske starosti od IV lunarnog meseca (LM IV) do LM X. Uzorci su podeljeni u 10 grupa prema gestacijskoj starosti. Primenom stereoloških metoda i korišenjem svetlosnog mikroskopa izraunata je volumenska gustina korpuskulske i tubulske komponente nefrona u korteksu humanog bubrega u razvoju. Rezultati. Polimorfizam
nefrona je glavna karakteristika bubrega humanog fetusa tokom razvoja. Kod mlaih fetusa neposredno ispod bubrežne kapsule nalazi se široka nefrogena zona.
Ona sadrži kondenzovan mezenhim i završne krajeve ureterskog pupoljka. Nefroni
u razliitom stadijumu razvoja su locirani oko ureterskog pupoljka koji se grana u
korteksnoj nefrogenoj zoni i indukuje nefrogenezu. Zreliji nefroni su locirani u dubljim partijama kore u blizini juksta-medulskog spoja. Tokom gestacije nefrogeneza
napreduje, a broj nefrona se poveava. Glomeruli menjaju veliinu i oblik, a menja
se i dužina i izvijuganost tubula. Bubrežna kora postaje šira i sadrži sve zrelije glomerule i vijugave tubule. Volumenska gustina tubulske komponente nefrona raste
neprekidno od 10,53% (LM IVa) do 27,7% (LM X). Renalni korpuskuli iregularno
menjaju vrednost volumenske gustine tokom gestacije, pokazujui porast od 13%
(LM IVa) do 15,5% (LM IVb). Porastom gestacijske starosti volumenska gustina
korpuskulske komponente nefrona se smanjuje do 11,7% (LM VIII), a zatim ponovo
poveava sve do kraja intrauterusnog razvoja (LM X) kada korpuskuli zauzimaju
16,73% zapremine bubrežnog korteksa. Volumenska gustina nefrona u razvoju
(korpuskulske i tubulske porcije) pokazuje statistiki znaajnu pozitivnu korelaciju
sa gestacijskom starošu (r = 0,85; p < 0,01). Zakljuak. Ova studija je jedna od
nekoliko koje kvantifikuju razvoj nefrona kod oveka. Poznavanje normalnog razvoja humanog bubrega može biti od velikog znaaja za buduu kliniku praksu.
K lj u n e r e i :
embrion i fetus, razvoj; bubreg; nefroni.
Correspondence to: Marija Dakovi-Bjelakovi, Department of Anatomy, Medical Faculty, University of Niš, Brae Taskovia 81, 18000 Niš, Serbia and montenegro, Tel. +381 18 532381