Quantitative analysis of the nephron during human fetal kidney development

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.


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 5 th week of intrauterine life.From the 8 th to the 36 th weeks of gestational age, nephrogenesis is continually induced by the ureteric bud ramifications (4,5).The nephron progresses through four devel-ɋɬɪɚɧɚ 282 ȼɈȳɇɈɋȺɇɂɌȿɌɋɄɂ ɉɊȿȽɅȿȾ Ȼɪɨʁ 4 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.

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 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 M 42 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).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).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.

ub cm
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. 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).

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 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 40 th 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.

Fig. 1
Fig.1Light micrography of human fetal kidney(13 th  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

Fig. 4
Fig. 4 Light micrography of part of the cortex in human fetal kidney (23 rd week of gestation).HE, u 100