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Journal of the Anatomical Society of India

Morphological and Morpho-metrical Study of Human Renal Development During Mid-Gestation Period

Author(s): Sabita Mishra, Anant Dinesh, J. M. Kaul

Vol. 55, No. 2 (2006-07 - 2006-12)

Sabita Mishra, Anant Dinesh, J. M. Kaul
Maulana Azad Medical College, New Delhi

Summary:

With the advent of modern technology and equipments for diagnostic and therapeutic procedures in the field of medical sciences it becomes imperative to have sound knowledge of the basic human morphology and developmental anatomy. The question of human renal development stages the same outcome with increasing emphasis on prenatal diagnosis of congenital malformations. There are few reports regarding the morphological development of human kidney and hardly any work considering both morphological and stereological aspect of development. So, our study aims at contributing to the knowledge of human renal development during mid-gestation period ranging from 14th to 28th week considering both qualitative and quantitative parameters. The right kidney of 15 human foetuses were dissected and processed in paraffin, 7mm thick sections were stained with Haematoxylin-Eosin and Mason’s Trichrome stains. The qualitative observations were carried out under a binocular light microscope while some stereological parameters like total renal volume, glomerular density and glomerular area, were measured using an interactive image analyzer. The volume of kidney showed a linear growth from 14th week to 28th week of gestation with growth spurts during 22nd and 28th week. The glomerular density exhibited a fluctuating trend with abrupt fall during 16th, 22nd and 28th week. The glomerular area showed an initial increase up to 16-18 weeks and then a gradual fall to reach an average around 6000-7000 sq. micron. The present work made an initial attempt to compare both qualitative and quantitative aspects of human renal development and to analyze the growth pattern of kidney in human fetuses.

Keywords: Kidney, Morphology, Morphometry, Development.

Introduction:

The emphasis on genetic counseling and the possibility of early prenatal diagnosis of congenital malformation have stimulated interest in fetal anatomy (Sampaio 1990). There are few reports regarding the morphological development of human kidney and hardly any work considering both morphological and stereological aspect of development. The present study aims at contributing to the knowledge of human renal development during mid-gestation period ranging from 14th to 28th week considering both qualitative and quantitative parameters. Previous studies have determined the morphological changes and a few stereological parameters with regard to the developing kidney (Almeida et al 2002).

The definitive human kidney is derived from an evagination of mesonephric duct (ureteric bud) and metanephric blastema with local mesenchyme giving rise to angiogenetic component. The angiogenic mesenchyme migrates into metanephric blastema to form glomeruli and vasa recta (Sadler; 2004; Cerrian et al 2004, Bannister 1995, Hamilton 1972, Osathanondh and Potter 1963, a, b, c)

In brief, the ureteral bud developing from the mesonephric duct dilates to form the ampulla which induces the surrounding mesenchymatous tissue i.e. metanephros resulting in the differentiation of mesenchymal agglomerates and their organization in concentrates. The condensate then epithelises and forms vesicles, which fuses with the ampulla to form a nephron. First vesicle is formed at the end of 7th week in relation to 6th division of ureteric bud. Cells at the proximal pole of the vesicles organizes to form C-shaped or comma shaped body followed by cellular reorganization of tubular cells at distal end to form a Sshaped body or S-body. In the cleft of the S-body at the distal pole, formation of extra cellular matrix and penetration by capillaries targets at formation of future mesangial region. The proximal limb of the S-body organizes to form distal convoluted tubule while the intermediate limb enlarges to form loop of Henle and the proximal convoluted tubule resulting in entire development of a nephron. Many such nephrons are present in the fetal kidney due to multiple branching of the ampullary bud and induction of various mesenchymatous condensates to form nephron arcades. This process of renal development begins at deeper regions and reaches the peripheral part of the cortex with the advancement of ampulla in that region and terminates during the last month of gestation with subsequent interstitial growth (Almeida 2002, Osathanondh and Potter 1963, a, b, c).

Kidney size is presumably influenced both by genetic and environmental factors. The number of glomeruli at birth is presumably genetically determined. The size of kidneys is dependent on the number and size of nephrons (Potter et al 1943; Hinchliffe et al 1991; Nyengaard et al.1992). The total filtration surface area depends on the glomerular density and the glomerular surface area, any variation in these factors alters the total filtration area which is a useful indicator of renal development.

The estimation of renal glomerular volume is a useful technique with clinical, diagnostic and prognostic relevance in several conditions including renal artery stenosis, glomerulosclerosis and glomerulomegaly (Beech 2000), Some authors have presumed that the retardation of renal development, as occurs in individuals of low birth weight, gives rise to increased postnatal risk of systemic and glomerular hypertension, as well as enhanced risk of expression of renal diseases such as aplasia, hypoplasia, cystic diseases or renal agenesis (Behrman. 2004, Almeida et al 2002)

Manifestations of renal disorders are directly related to developmental anatomy of kidney including both morphological and stereological parameters (Behrman 2004). Thus, in utero detection of anomalies prevents delay in postnatal diagnosis and enables early surgical repair of significant lesions (Sampaio and Aragao 1990; Sampaio 1992).

Thus, the present work aims to provide morphological development of kidney in human fetuses and stereological estimation of the total volume of kidney, glomerular density, glomerular area. Since there are very few human studies in regard to the developing kidney the present study was conducted.

Material and Method:

Fifteen aborted human fetuses were collected from labor room of Lok Nayak Jai Prakash Hospital (LNJP), New Delhi after the consent of parents, following all ethical norms and clearances. Fetuses were immediately immersed fixed in 10% formalin. All were in excellent condition of preservation and none of them had any congenital malformation. Gestational age was determined in weeks post conception (WPC) according to Crown Rump Length (CRL), Biparietal Diameter (BPD) and Foot length criterion (Hamilton 1972) and ranged from 14 to 28 weeks. The right kidneys of fetuses were dissected out and fixed in freshly prepared 10% formalin for a minimum of 3 days. The entire kidneys were embedded en block in paraffin using the standard protocol. Initially the physical dimensions of each kidney were measured using a linear scale, a divider and thread. Tissue blocks were serially sectioned in a transverse plane parallel to the long axis of the kidney to generate 7μm thick sections, using a rotary microtome. The sections after mounting were stained with Haematoxylin- Eosin and Mason’s Trichrome stain.

Morphological Analysis

The histological sections were studied using a Labmed binocular light microscope under 4X, 10X and 40 X objective lenses with the aid of an interactive image analyzer.

Stereological Procedures

Estimation of glomerular area

Glomerular area for each kidney at different gestational ages was estimated by measuring the area of 100 glomeruli per fetal kidney under 40 X objective lens of the image analyzer (where 1 pixel=1.39 m) with the aid of the software package Dewinters Biowizard 2.0.

Estimation of numerical density (ND) of glomeruli in the cortex

The numerical density of glomeruli in the cortex of each kidney at different gestational ages was estimated by observing 10 microscopic fields per fetus under 10 X objective of image analyzer (where 1 pixel=5.49 m) with the aid of a specific software package Dewinters Biowizard 2.0.

The microscopic field area (A) was kept constant and the counting was done for all fields using the grids to reduce counting errors. The average particle count (nav) was estimated and numerical density was calculated using the formula.

Estimation of Total Renal Volume (Vtotal)

The total volumes of each kidney were estimated using a Cavalieri’s Principle. Consecutively 5th sections were projected onto a screen using a slide projector. The image was superimposed on a square grid with each square measuring 1 mm2.

The renal volume was estimated by formula:

Observations:

Morphological Observations

At 14 weeks of gestation kidney was in a lobulated form showing few differentiated lobules and there were some lobules showing undifferentiated mesenchyme. Hilar region had abundance of mesenchyme in which the hilar vessels and developing pelvis was observed (Fig 1). These differentiating lobules showed C-shaped and S-shaped developing tubules and presence of a few developing glomeruli (Fig 2). At higher magnification metanephric blastema was seen near the developing glomeruli (Fig 3).

Figure 1: Depicts at 14 weeks of gestation lob1ular organization of metanephric blastema (2 lobules seen). Developing excretory duct, the future pelvis (P) and Differentiating blood vessels (BV) between the lobules. Magnification 4x. Haematoxylin and Eosin stain.

Figure 2: Presence of peripherail neuphrroecoele (*) and differentiating nephrons (arrow) during 14 weeks of gestation Magnification 10x. Haematoxylin and Eosin stain.

Figure 3: Depiction of matanephigric blarsetemal cap (arrow) over the developing ampullary bud during 14 weeks of gestation Magnification 40x. Haematoxylin and Eosin stain.

At 16 weeks of gestation, the kidney showed evidence of lobulation but lobules appeared fused with each other. A zone of transition appeared between the cortex and medulla of the developing kidney. The peripheral part of cortex was deeply stained indicating increased activity while medulla still showed mesenchymatous tissue with some developing tubules in it. Juxta-medullary glomeruli were larger in size while the peripheral ones appeared smaller. By 18-20 weeks of gestation cortico-medullary junction was well defined. Juxta-medullary glomeruli appeared larger with large number of tubules. There was a well-defined arcade system and organization of collecting ducts was observed. There was also presence of pyramids opening into excretory part with collecting ducts converging onto it (Fig 6). Some of the tubules appeared stratified (Fig 4). The renal pelvis was lined by transitional epithelium with ampulla having undifferentiated mesenchyme (Fig 5).

Figure 4: A section showing stratification of tubes during 18 weeks of gestation. Magnification 40x. Haematoxylin and Eosin stain

Figure 5: Depicts successive generation of ampulla (A) at prospective renal pelvis (P) in a fetal kidney at 20 weeks of gestation Magnification 4x. Haematoxylin and Eosin stain

Figure 6: Orientation of collecting ducts (arrow) at 20 weeks of gestation. Clear demarcation between cortex and medulla observed Magnification 4x. Haematoxylin and Eosin stain

At 22 weeks of gestation the cortex and medulla appeared to be much more mature. The glomeruli were well formed. At higher magnification two types of tubules with different staining pattern one was deeply eosinophillic and the other was deeply basophilic. The deeply eosinophillic ducts were smaller with round nucleus and larger lumen (Fig 7). At 24 weeks of gestation there was a clear demarcation between cortex and medulla the cortex was thicker and the juxtamedullary glomeruli were larger arranged in 2-3 rows. Collecting ducts and the loop of Henle appeared elongated. There were some ampullary arcades at the juxta cortical area (Fig 8). At 28 weeks of gestation well-defined pyramid system was delineated. The collecting ducts were seen converging onto the excretory system. Evidence of mesenchymatous tissue at the periphery was still present (Fig 9).

Figure 7: Differentiation of 2 sets of tubules deeply eosinophilic (pink arrow) and deeply basophilic blue arrow during 22nd week of gestation Magnification 40x. Haematoxylin and Eosin stain

Figure 8: Well defined glomeruili and tubeules seen at 24th weeks of gestation Magnification 10x. Massons Trichrome stain.

Figure 9: Well defined diffeFrenitgiatiuonr beetween cortex and medula during 28 weeks of gestation. Collection ducts seen converging towards the excretory system (arrows) Magnification 10x. Haematoxylin and Eosin stain.

Stereological Observations

The volume of kidney showed a linear growth from 14th week to 28th week of gestation with growth spurts during 22nd and 28th week (Graph I). The glomerular density exhibits a fluctuating trend with abrupt fall during 16th, 22nd and 28th week (Graph I). The glomerular area showed an initial increase up-to 16-18 weeks and then a gradual fall to reach an average around 6000-7000 sq. micron (Graph II).

Discussion:

The present study illustrates a sequential view of human renal development in light microscopy, both qualitatively and quantitatively, from mesenchymal concentrates to well-defined structures, still under the process of further differentiation. Morphogenesis of human kidney begins at 5th week of gestation and extends upto the last month of third trimester of pregnancy (Cerrian C, 2004, Hamilton 1972). There have been some studies regarding the glomerulogenesis in human fetal kidney. Most of the results in the present study are in accordance with the previous authors while some interesting findings, which still remain unquoted, have been delineated.

The morphological study outlines the presence of lobulation at 14 weeks in utero, while at 16 weeks the kidney fused to form a single organ though the lobulation was still evident, which is in agreement with previous authors (Osathanondh and Potter 1963 a,b,c). In the current study, the development of tubules to C-shaped and S-shaped bodies was observed, as described by various workers (Almedia 2002, Hamilton 1972). The differentiating glomeruli, cortico-medullary junction and the formation of renal pyramids was also observed as described previously (Cerrian et al 2004, Hamilton 1972) Development of well-defined arcade system during 18 weeks of gestation and degeneration of some glomeruli with increase in age lies in accordance with previous observations made by (Osathanondh and Potter 1963 a,b,c). An important observation of difference in staining pattern of tubules at 22 weeks in utero, finds no mention before. Two types of tubules were seen, one being deeply basophillic and other eosinophillic with larger lumen and round nucleus which is difficult to interpret at this stage whether they were proximal or distal convoluted tubules. It can only suggest that probably there is a change in the pH of urine at this age. Though the standard textbooks mention urine to be hypotonic in utero (Nyengaard et al 1992).

The stereological results presented an explicable pattern. Glomerular density from 14th to 28th week of gestation showed abrupt rise and fall which could be easily explained by the sudden increase in volume of kidney from 22nd to 28th weeks and variation in mean glomerular size with increasing gestational ages (Osathanondh and Potter 1963; Sampaio 1990, Hinchliffe et al 1991). The pattern of growth in kidney volume and glomerular sizes followed the same trend as observed by previous authors (Fujikara and Froehlich 1972, Nyengaard et al 1992). Surprisingly at 28th week, the glomerular area shows a significant decrease. The decrease in the size of the glomeruli could be because of the reorganization in the following manner. The newer glomeruli which are smaller are being pushed towards the periphery and in this study the total cortical glomeruli have been taken into consideration, thereby showing a significant decrease in the glomerular area. The study includes the morphological and stereological results of only right kidney as the statistical analysis previously made, states no difference between the growth of right and left kidney volumes, for male or for female fetuses (Behrman 2004), while there is a difference in fetal kidney length growth between male and female fetuses (Sampaio 1990), which holds no significance in present study.

The present work made an initial attempt to compare both qualitative and quantitative aspects of human renal development and to analyze the growth pattern of kidney in human fetuses, which may prove useful in defining the fetal kidney diseases such as agenesis, hypoplasia, multicystic kidney, polycystic kidney etc., more precisely using the modern invasive or non-invasive imaging technique, Therefore, from the current study one can conclude that major part of development of fetal kidney occurs during mid-gestation period and continues until the last week of third trimester of gestation.

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