Evaluation of absolute volume of human fetal kidney ' s cortex and medulla during gestation

Background. Human fetal kidney is quite different from the mature kidney, both macroscopically and hystologically. Lobulated surface of the human fetal kidney reflects its inner organisation. Aim. To determine the fetal kidneys' volume according to the gestational age, to establish periods of their maximal and minimal growth and to compare these values for various gestational ages. Methods. Forty five human fetal kidneys aged from IV to X lunar months were analyzed. Kidneys were divided into nine groups according to their gestational age. The volumes of cortex and medulla were determined using stereological methods. The results were statistically analyzed and the periods of significant growth of these structures were marked. Results. Fetal kidney's cortex and medulla grew continually with a very high coefficient of linear correlation with crown-rump length. The cortex/medulla ratio was minimal in the first half of V lunar month, when medulla grew most rapidly and it was maximal immediately before birth, when cortex achieved its maximum. Conclusion. This study was an effort to provide some parameters which would help in the future investigations of the development of human fetal kidney.


Introduction
Interest in fetus' life used to be focused, for centuries, on the studies of embryo and mechanical problems encountered by gynecologists at the delivery.Nowadays, it is well known that the development of fetus is not proportional to the growth of its organs, and that each stage in fetus' life is specific (1,2).
The development of human fetal kidney goes through a series of continual and mutually dependent changes during which it reaches both morphological and functional maturity.The existing literature suggested that stereological and quantitative methods dealt with the adult kidneys much more than with the fetal ones.Many papers discussed the use of stereological method for the human bioptic material, donor's kidney during transplantation, material obtained during operations, cadaveric material, etc (3).There are also more papers on the kidneys of experimental animals than on the human fetal kidneys, as well as on stereology.Quantitative data and spatial consideration of kidney's structure is more important nowadays, because of the more frequent use of the early intrauterine diagnostics of urinary system disor-ders and the use of intrauterine surgery procedures (4í6).Stereological researches on the human fetal kidney are important for better understanding of the normal development of its structure.It is neccessary to study the fetal kidney, especially its productive part, parenchyme, as well as its function during the fetal life.Knowledge about normally developed structures of the fetal kidney may have a decisive influence on planning of the procedures for high-risk pregnancy and the prevention of renal insufficiency.
The aim of this research was to review and quantify the changes in kidney's parenchyme (cortex and medulla) within the fetal period of development, as well as to determine a quantitative cortex/medulla ratio due to their different functions.

Methods
In this study, 45 fetuses were analyzed, divided according to the lunar months and based upon temple to crown-rump length (CRL (7).According to the literature, preliminary research and intensive growth of the examined structural parts of the kidney at IV and V lunar month were ɋɬɪɚɧɚ 108 ȼɈȳɇɈɋȺɇɂɌȿɌɋɄɂ ɉɊȿȽɅȿȾ Ȼɪɨʁ 2 divided into two subgroups, the first and the second half.All fetuses were fixed in 10% formaldehyde for at least 30 days.Macrodissection was used to extract both kidneys en block and separate them from the surrounding tissue.For stereological analysis, two test systems were used.For the kidneys at IV, V and VI month, D64 system was used, and for the kidneys at VII, VIII, IX and X month, M42 system was used.Different test systems were used because of the different-sized cuts.D64 system has more points than M42 system rendering it more suitable for smaller fetal kidneys.As a result of this fact, approximately equal number of the points fell on the surface of each cut.Test systems were photocopied to transparent paper and reduced to the size suitable for analysis.Using micrometer, the distance d was measured, while the characteristics of testing systems were established by general formulae.The kidneys were cut at an angle of 90° to their axis at the distance of t = 2.5 mm for IV, V and of VI month and t = 5 mm for VII, VIII, IX and X month.The cuts revealed clearly the renal sinus and parenchyme rendering cortex and medulla easier to distinguish by the color.Testing system was directly placed on the surface of a cut allowing the points which fell on the cortex and medulla of the kidney to be counted, using microscope or magnifying glass (8) (Figure 1).The absolute volumes of kidney's cortex and medulla were obtained by Cavalieri method, usinga formula: Vm' = Pm a t, in which "Pm" is the number of points falling on the cortex or medulla, "a" the surface of test system which belongs to one point and "t" the distance between cuts (6, 9í11).
The obtained numerical values were corrected by coeficient of tissue contraction (shrinkage factor) to C = 1.24.All parameters were statistically analyzed and graphically shown (12).For statistical analysis, Student's t-test was used.The relation between the volume of fetal kidney's cortex and medulla and CRL was estimated using Pearson's correlation coefficient.

Results
The average volume of the left kidney's cortex continually increased from 226.5 mm 3 in the first half of IV lunar month up to 5 298.22 mm 3 at X lunar month (Table 1).The cortex of the left kidney showed statistically significant increase in the second half of V lunar month (p<0.05), at VI month (p<0.01), at VII month (p<0.01) and at VIII lunar month (p<0.05) with a very high correlation with CRL (r=0.972;p<0.01) (Figure 2).The average volume of the right kidney's cortex increased from 237.01 mm 3 up to 5 909.05 mm 3 at X lunar month (Table 1).Significant increase of the right kidney's cortex took place in the second half of IV lunar month (p<0.05), at VI, VII, VIII and IX (p<0.05), and at X lunar month (p<0.01).Correlation coefficient was very high (r = 0.947; p<0.01) (Figure 3).The previous data show that the cortex grew most rapidly from VI lunar month to the birth.
The average volume of fetal kidney's medulla is shown in Table 2.Both kidneys' growth correlated very well with CRL, the left one r = 0.994; p<0.01 and the right one r = 0.997; p<0.01 (Figures 4í5).In the left kidney, it increased from 176.15 mm 3 in the first half of IV up to 2 979.6 mm 3 in X lunar month.The medulla of the right kidney grew from 205.67 mm 3 in the first half of IV month up to 2 863.63 mm 3 at X lunar month.Asterisks in Table 2 indicate statistically significant increase in the medulla's volume as compared to the previous period.The kidney's medulla grew most rapidly between V and VII lunar month.
A cortex/medulla ratio was the lowest in the first half of V lunar month, showing a tendency to increase up to X lunar month when it reached its highest values.Unexpectedly, at IV lunar month it was larger than at V month (Table 3).

Discussion
As mentioned above, the fastest growth of renal cortex of both fetal kidneys was at VI, VII and VIII lunar month, namely, from 21st to 32nd week of the intrauterine life.The values obtained by this research were by 10í15% higher than those obtained by Hincliffe et al. who had studied a much smaller number of fetal kidneys (11 fetuses) (10).Significant increase in cortical volume in the later fetal age could be explained by the intensive nephronogenesis in the superficial cortex, as it is known that nephrons mature and grow centrifugally, i.e., toward the fibrous capsule (13,14).In accordance with these data, Dunnill and Halley established that the highest percentage of cortical volume belonged to glomeruli of the new-born kidneys (15).Thus, we could suppose that the cortical volume of the later fetal age increased mainly because of the increase in both number and size of the glomeruli and the elongation of the cortical tortuous tubules (16).
The absolute volume of the fetal kidney medulla increased with aging of a fetus, highly correlating with CRL.Kidney's medulla showed significant growth at V and VII month of gestation.The average values of medulla's volume established by this study were higher than those obtained by Hincliffe et al. (10).Considerable growth of kidney's medulla at the midgestation fetal age, mentioned in this study, could be the result of the extreme growth of juxtamedullar nephrons and their straight parts contrary to the growth of the cortex in the later fetal age (17).All of the above might have very interesting implications to the fetal renal function.The control of liquid volume and the retention of electrolytes depened mainly on the medullary parts of nephron, while filtration, selective secretion and reabsorption depened on the cortical parts of nephron (18).
A cortical/medullar volume ratio was related to the previous data.ýuš (19) cited the conclusions of Hollatz and Parade, who had investigated cortex/medulla ratio of infant, adult and mammalian kidneys.They concluded that this ratio was the lowest in a new-born, and that it continually increased up to the second decade of life.Next, it stagnated for a very long time and, finally, after 70 years it began to decrease.According to our results, this ratio increased from the second half of V lunar month of antenatal life and all the way to the birth.This tendency went on till the second decade of the postnatal life (18), without any changes immediately before or after birth.This fact showed clearly that the development of the organism began from the conception, not from the birth.
The results of these investigations could be used as a reference for the determination of some absolute values such as: absolute volume of the glomeruli, absolute number of the renal nephrons etc, when it was possible to obtain both numerical and volume densities of various kidney's structures out of a sample tissue.It could also be recommended to use an indirect method to the same purpose (11).This method is practical and quick for obtaining the absolute values which are significant for any stereological research.

Fig. 1 í
Fig. 1 í Test system M42 placed on the cross-section of fetal kidney.