Impairment of nephrogenesis
A series of noxae different in molecular compositions can provoke an early termination of nephron formation in preterm and low birth weight babies [1, 2]. While the clinical parameters have been intensely analyzed, comparatively little information is available with regards to the affected stages of nephron anlage, the concrete cell targets of noxae, and the molecular processes leading to pathological alterations in the outer cortex of the fetal human kidney. The situation is further complicated by the fact that the microscopic assessment of specimens needs special attention, since the target region of noxae is complexly built up. Covered by the renal capsule, it consists of fetal, maturing, and matured tissue layers [3]. This reflects a structural gradient, in which the process of nephron formation is integrated. It starts near the inner side of the renal capsule, lines perpendicular to it through the external nephrogenic zone, the subjacent maturation zone, and then the matured zone.
Detection of long-term effects
The transiently appearing stages of nephron anlage, which are restricted to the nephrogenic zone, are relevant for the impairment of nephrogenesis. Beginning with the nephrogenic niche, these include the successively developing pretubular aggregate, renal vesicles, and comma- and S-shaped bodies. Using the rodent kidney as an experimental model, it was demonstrated that the exposure to noxae targets the nephrogenic niche resulting in a low expression of morphogenic molecules such as Gfrα1, Gdnf, Bmp4, Pax2, and Six2 [4]. Surprisingly, comparable data dealing with a damage on the early stages of nephron anlage in the fetal human kidney is lacking. Instead, the limited data available shows that the width of the nephrogenic zone in gestational controls is 150 µm, while in preterm babies, it is significantly smaller at 100 µm [5]. Furthermore, the lack of basophilic S-shaped bodies in the nephrogenic zone of preterm babies was reported [6]. However, in these publications, information dealing with the pathological alterations of the tissues covering the starting nephron is not provided.
Frequently mentioned is the detection of atypical glomeruli including a dilated Bowman’s space and a shrunken glomerular taft [5] and/or a decrease in the number of glomeruli [7]. However, these pathological findings do not concern primarily the stages of nephron anlage in the nephrogenic zone but the maturing nephron in the underlying maturation zone. Surprisingly, no previous related publications provide a clear proof on whether the noxae impairing nephrogenesis has a direct impact on the developing nephron, or whether it is indirect and caused by a disturbance of the covering tissues and related molecular interactions.
Rather unexplored terrain
Although surprisingly, the analysis of pertinent literature further revealed that the role of the nephrogenic zone in the fetal human kidney in nephron formation and consequently as the primary target region for noxae impairing nephrogenesis has been hardly explored. Before this background, it is understandable that the contained stages of nephron anlage and the shaping of the nephron were only introduced over the past few years as the first systematic morphological information dealing with the specifics of the nephrogenic zone [8,9,10]. The results generated indicate that each stage of nephron anlage is the subject of an individual positioning, orientation, and shaping. A further important finding in this context is that during the progress of development, not only the individual stage of nephron anlage but also the corresponding covering tissues are specifically changing. As a consequence, the aim of the present morphological investigation is to demonstrate the precise site of nephron formation, to inform about important coordinates, and to document the mutual patterning of its covering tissues.
Zones in the outer cortex
Noxae impairing nephrogenesis targets the outer cortex of the fetal human kidney during late gestation. Covered by the renal capsule, it is built up by transverse layers of embryonic, maturing, and matured parenchyma and stroma. In the external nephrogenic zone, the process of nephron formation starts [9] and can be recognized by the transient stages of nephron anlage [10, 11]. In the subjacent maturation zone, the conversion of the S-shaped body as the last stage of nephron anlage into the lasting nephron occurs. This process comprises the elongation, spatial extension, and functional differentiation of the individual nephron segments. In the underlying matured zone, the typical morphological features of the definitive nephron are established.
Location of initial nephron morphogenesis
Most relevant in the search for initial imprints left by the impairment of nephrogenesis is the external nephrogenic zone. This can be seen underneath the renal capsule as a thin strip of embryonic parenchyma and stroma [9, 12]. In the fetal human kidney, the outer border is in contact with the inner side of the renal capsule. The inner border has been previously defined as a “dotted” line, which meets the proximal (medulla-orientated) pole of the side by side aligned S-shaped bodies in a transverse orientation [5, 13]. Furthermore, it has been defined that the vertical distance between the renal capsule and the proximal pole of an S-shaped body is 150 µm. At its inner border, the nephrogenic zone is facing the subjacent maturation zone. Components of the nephrogenic zone are the nephrogenic and stromal progenitor cells, the transiently appearing stages of nephron anlage such as the nephrogenic niche, pretubular aggregate, renal vesicles, and comma- and S-shaped bodies. In addition, the ureteric bud-derived collecting duct (CD) ampullae are extending at the distal endings of the collecting duct tubules. The perforating radiate arteries line in vertical direction. In the arising interstitium and along the microvascular system, numerous macrophages are visible [14].
Individual site of nephron formation
When vertical lines are drawn, the nephrogenic zone can be divided into numerous nephrogenic compartments [10, 11, 15]. These are aligned side by side in a row. Within a nephrogenic compartment, the development of a single nephron from the recruitment of progenitor cells to the late S-shaped body takes place (Fig. 1). At the top, each of the nephrogenic compartments is covered by the renal capsule. Its medial border can be recognized by a vertically lining CD ampulla. The lateral border is defined by a perforating radiate artery, which lines vertically towards the renal capsule. The border at the base is the proximal pole of the respective stage of nephron anlage up to the S-shaped body. This is positioned above the connecting tubule of an earlier developed nephron.
Within a nephrogenic compartment, a certain number of progenitor cells of both the presently developing nephron and the future generation are produced. This not only requires the number of progenitor cells to be controlled, but also to ensure that they remain in the correct place and that a percentage is transferred into the state of competence for induction [16]. After the exchange of morphogenic molecules in the nephrogenic niche, the actual morphogenesis can begin. This stage can be recognized by a cell aggregation and the subsequent formation of the pretubular aggregate (Fig. 1a), the mesenchymal to epithelial transition (Fig. 1b), and the development of the primitive renal vesicle (Fig. 1b) next to the tip and then the head of the CD ampulla. When the mature renal vesicle separates from the pretubular aggregate (Fig. 1c), in order to begin with the shaping of the nephron (Fig. 1d–l), the backdrop of development is shifting to the conus of the CD ampulla. For this reason, it is important to differentiate between the above-positioned district of progenitor cell recruitment and the underlying area of nephron shaping (Fig. 2).
District of progenitor cell recruitment
The district of progenitor cell recruitment represents a prone rectangle, which principally maintains its size during the formation of a nephron (Fig. 2a) [10, 11]. It is in direct contact with the inner side of the renal capsule and shows a transverse length of about 80 µm. The medial and the lateral borders are 50 µm in length on average. Its lower transverse border lines through the section border between the head and conus of the related CD ampulla.
The tissue configuration in the district of progenitor cell recruitment is remarkable. When histological sections of the pretubular aggregate are observed, the available space between the tip of a CD ampulla and the inner side of the renal capsule is surprisingly narrow (Figs. 1a and 2a). Only 2 to 3 layers of mesenchymal progenitor cells are seen here [8]. For the rodent kidney, it was shown that these layers are arranged in a topological order. The nephrogenic progenitors face the tip of a CD ampulla, while the stromal progenitors are distributed near the inner side of the renal capsule [17].
At the tip of a CD ampulla, the nephrogenic niche is formed as the first stage of nephron anlage. In the immediate vicinity, as well the aggregation of induced nephrogenic mesenchymal cells, the formation of the pretubular aggregate (Figs. 1a and 2a), the mesenchymal to epithelial transition (Figs. 1b and 2b), and the appearance of the primitive renal vesicle (Figs. 1c and 2c) is noticed. The section border between the head and the conus of the CD ampulla determines the further process. Here, a link with the primitive renal vesicle takes place to fix the future connecting tubule (CNT). During this process, at the medial part of its distal pole, the renal vesicle is separated from the pretubular aggregate, while the lateral part remains connected via a two-layered progenitor cell strand (Figs. 1c, d and 2c, d).
Area of nephron shaping
This area represents an extending quadrate, in which the development from the mature renal vesicle (Figs. 1c and 2c) to the late S-shaped body (Figs. 1l and 2l) is successively progressing. At the beginning, the area of nephron shaping resembles the small size of a mature renal vesicle (ca 20 × 20 µm), but finally reaches vertical and transverse lengths of up to 100 µm each during the development of the S-shaped body [10, 11].
The upper border of the area of nephron shaping lines transversely at the section border between the head and conus of the related CD ampulla. It also meets the attachment site of the mature renal vesicle on the CD ampulla, as the proximal end of the pretubular aggregate (Figs. 1d and 2d). More laterally, the upper border crosses the mesenchymal progenitor cell strand, which connects the proximal end of the pretubular aggregate with the extending (Figs. 1e and 2e) and extended (Fig. 2e, f) renal vesicles. In the early comma-shaped body, it crosses the site where the separation from the pretubular aggregate occurs (Figs. 1g and 2g). The medial border lines along the conus of the CD ampulla, while the lateral border is near a vertically lining perforating radiate artery. The lower transverse border lies between the external nephrogenic zone and the subjacent maturation zone. As a consequence, it crosses the proximal pole as well as that of the developing renal vesicles (Figs. 1d–f and 2d–f) and the comma- (Figs. 1g–i and 2g–i) and the S-shaped bodies (Figs. 1j–l and 2j–l). As previously mentioned, the respective proximal pole rests near the transversely lining connecting tubule of an earlier developed nephron. The configuration resembles an immovable base, which blocks the spatial expansion of the developing nephron near the proximal pole, but enables the lifting of the entire nephrogenic compartment step by step together with the renal capsule. Finally, regarding microscopic specimens within the area of nephron shaping, beside the attachment site of the future connecting tubule (CNT) at the section border between the head and conus of the CD ampulla either mature, extending, or extended renal vesicles (Figs. 1d–f and 2d–f), a comma-shaped body (Figs. 1g–i and 2g–i) or a S-shaped body (Figs. 1j–l and 2j–l) can be observed.
Contours at the transient stages of nephron anlage
For the fetal human kidney during late gestation, it was demonstrated that the initial formation of a nephron takes place in a nephrogenic compartment located in the nephrogenic zone [10]. In microscopic specimens, this is recognized by the transient stages of nephron anlage including the nephrogenic niche, pretubular aggregate, renal vesicles, and comma- and S-shaped bodies, which are present at this point (Figs. 1 and 3).
Nephrogenic niche, pretubular aggregate, and primitive renal vesicle
The formation of a nephron is initiated by the process of induction. This takes place in a nephrogenic niche and within close proximity to the inner side of the renal capsule [8]. During this process, the most inner layer of the nephrogenic progenitor cells faces the basal aspect of the epithelial progenitor cells contained in the ureteric bud-derived tip of a CD ampulla. A series of morphogenic molecules such as Gdnf, Wnts, Fgfs, and Bmps are then exchanged [16]. It can be recognized that in this situation, the nephrogenic mesenchymal progenitor cells are separated from the tip of the CD ampulla by a clear interface (Figs. 1a and 3a) [10].
When the process of induction is successful, the nephrogenic mesenchymal progenitor cells increase in size, their shape becomes angular, and the intercellular spaces enlarge to aggregate firstly along the tip and then head of the CD ampulla. This leads to the formation of the pretubular aggregate, which looks like a teardrop (Figs. 1a and 3a). Its thin distal end is orientated towards the renal capsule so that it remains in contact with the inner layer of the nephrogenic mesenchymal progenitor cells. In contrast, the thickened proximal end points towards the medulla. A clear interface is visible between the distal end of the pretubular aggregate and the tip of the CD ampulla. However, between the proximal end of the pretubular aggregate and the head of the CD ampulla, a close adhesion is noticed. Uneven interstices between the cells and a smoothening of the surface at the proximal end of the pretubular aggregate are first signs for the mesenchymal to epithelial transition (MET) (Figs. 1b and 3b).
As a result, the primitive renal vesicle, with a polarized epithelium and a small lumen, becomes visible (Figs. 1c and 3c). In parallel, a transverse but incomplete separation of the renal vesicle from the pretubular aggregate is seen at the border between the head and conus of the CD ampulla. The process of separation extends transversely as far as the middle of the pretubular aggregate, so that the lateral part of the renal vesicle remains connected at this timepoint with the pretubular aggregate via a two-layered progenitor cell strand.
Mature, extending, and extended renal vesicles
After the development of the primitive renal vesicle, the setting is translocated from the district of progenitor cell recruitment to the underlying area of nephron shaping (Figs. 1d–f and 3d–f). In the rodent kidney, a distinction is made between the primitive, mature, and extending renal vesicles 1 to 5 [17,18,19]. Comparable experimental information in the fetal human kidney is not currently available. However, on the microscopic specimens, a site of considerable experimental interest is the distal pole of the mature renal vesicle (Figs. 1d and 3d). Its medial part detaches from the pretubular aggregate to be fixed on the CD ampulla at the section border between its head and conus. At this site, the future connecting tubule (CNT) and the tubule anlage become visible. Noticeable, the inner part of the distal pole remains detached from the overlying pretubular aggregate, while the lateral part remains connected with the pretubular aggregate via the two-layered progenitor cell strand. Due to the epithelial folding, the lumen in a mature renal vesicle is uneven, and it is V-shaped at the distal pole and rounded off at the proximal pole. Between the conus of the CD ampulla and the medial aspect of the renal vesicle, a vertically lining interstitial cleft starts to develop.
In the extending renal vesicle, the lumen is changing (Figs. 1e and 3e). While remaining rounded at the proximal pole, the vertical elongation of the tubule anlage causes a protrusion at the distal pole. Following this, the renal vesicle continues to extend not only lengthwise but also widthwise. The interstitial cleft then begins to elongate vertically between the conus of the CD ampulla and the medial aspect of the renal vesicle.
In the extended renal vesicle, the future CNT invades the epithelium of the CD ampulla in order to connect with it at the section border between its head and conus (Figs. 1f and 3f). The interstital cleft between the conus of the CD ampulla and the medial aspect of the renal vesicle elongates vertically up to the CNT. The lateral part of the distal pole is still connected with the pretubular aggregate via a two-layered progenitor cell strand. The tubule anlage elongates in a vertical direction to form an inner fold. The medial leg lines up with the epithelial cell strand of the presumptive CNT to end at the head of the CD ampulla. The lateral leg of the fold is part of the progenitor cell strand, which is connected with the pretubular aggregate. An oblique interstitial cleft in form of a narrow pocket becomes visible.
Comma-shaped body
During the development of the early comma-shaped body, the two-layered mesenchymal progenitor cell strand between its distal pole and the overlying pretubular aggregate is dissolving (Figs. 1g and 3g) [10]. Henceforth, the developing nephron can no longer partake in the progressive recruitment of progenitor cells [20]. The tubule anlage elongates vertically towards the center of the comma-shaped body. This causes the medial, inner as lateral folds to elongate. As a result, the comma-shaped body expands in vertical direction. At its proximal pole, the glomerulus including the Bowman’s capsule develops histo-typical features.
During the formation of the mid comma-shaped body, the epithelium of the CNT fuses with the epithelium of the CD ampulla at the border between its head and conus (Figs. 1h and 3h). The interstitial cleft between the conus of the CD ampulla and the medial aspect of the comma-shaped body continues to extend in length. In addition, an elongation of the proximal tubule segment is observed. The visceral epithelial cell layer transforms into the podocytes.
In the late comma-shaped body, the tubule segments further extend by meandering (Figs. 1i and 3i). At the medial aspect, the cleft between the conus of the CD ampulla and the comma-shaped body changes direction by expanding along the CNT. At the lateral aspect, the cleft between the proximal tubule segment and the visceral epithelial cell layer including the podocytes is changing direction from vertical to transverse. Consequently, it opens laterally and in the close proximity of a vertically lining perforating radiate artery. This constellation enables an afferent arteriole to invade the developing glomerular tuft via a shortcut.
S-shaped body
The S-shaped body is the last stage of nephron anlage, which develops in the nephrogenic zone and has been analyzed in both the rodent and human kidney [21, 22]. The early S-shaped body still develops perpendicular to the renal capsule (Figs. 1j and 3j). At its distal pole, the CNT completes the connection with the CD ampulla at the section border between its head and conus, while at its proximal pole, the glomerulus and the Bowman’s capsule present typical morphological features. Both structures are separated from the tubular portion by a transversely lining interstitial cleft. Inside this cleft, the glomerular tuft, the intra- and extraglomerular mesangium as the capillary network are establishing themselves. It opens at the deep lateral aspect of the S-shaped body near a vertically lining perforate radiate artery.
At the medial aspect, the mid S-shaped body is separated from the conus and neck of the CD ampulla by a vertically lining interstitial cleft (Figs. 1k and 3k). It appears that the Bowman’s capsule and the conus of the CD ampulla form nearly congruent surfaces. At its proximal pole, the glomerulus extends causing the overlying interstitial cleft to elongate and change direction from vertical to transverse.
The late S-shaped body expands mainly in a vertical direction (Figs. 1l and 3l). At its proximal pole typical morphological features of the glomerulus are visible. The interstitial cleft between the developing glomerulus and the overlying tubular portion further extends so that a network of capillaries and the intraglomerular mesangium can establish. After reaching the interior of the S-shaped body it changes direction from transverse to sickle-shaped. In contrast, the interstitial cleft between the conus, and the neck of the CD ampulla and the medial aspect of the S-shaped body respectively, lines vertically to the CNT and then horizontally so that it is reaching finally the geometric center of the S-shaped body.