Differentiation of glomerular filter and tubular reabsorption apparatus during foetal development of the rat kidney

Differentiation of the nephron and particularly of the glomerulus has been investigated both by light and by electron microscopy (Montaldo & Piso, 1970; Leeson, 1961 ; Kasimierczak, 1971 ; Miyoshi, Fujita & Tokunaga, 1971 ; Crocker & Easterbrook, 1972; Potter, 1972; Zimmermann & Boseck, 1972; Larsson, 1975; Kasimierczak, 1976) but hitherto, no study has been performed on the correlations between the structural development of the protein retention system (glomerular filtration barrier and tubular reabsorption apparatus) and the onset of filtration and selective permeability properties.

Development of the metanephros begins during foetal life but is only completed after birth. All the nephrons do not develop at the same time. The first nephrogenic masses appear under the kidney capsule and are progressively differentiated and connected to the collecting tubules; new nephron generations are formed at the periphery of the kidney so that, at any time of foetal life, the most differentiated ones will be the most deeply embedded. At birth, most nephrons appear as mature formations but in certain species, particularly in the rat, some nephrogenic masses can still be seen, their differentiation being achieved several days or weeks following birth (Kasimierczak, 1971; Larsson 1975; Kasimierczak, 1976).

The purpose of this paper is to examine ultrastructural differentiation (especially with regard to the glomerular filtration barrier and proximal tubule endocytosis apparatus) and protein filtration-reabsorption properties of immature nephrons, at different stages of foetal life.

Sherman rats were used in all experiments. For foetal studies, two-month-old females weighing 200– 300 g were mated overnight, the following day being considered as day 0 of gestation.

Studies were performed on rat foetuses from day 15 to day 21 of gestation and on newborn, 3-day-old, 25-day-old and adult animals. Ten animals were used for each stage. Rats were anaesthetized intraperitoneally with sodium pentobarbital (6 mg/100 g of body weight). In all cases fixation was performed by vascular perfusion. It was essential to prevent disruption of blood perfusion and vascular hypertension to avoid structural disturbances (Maunsbach, 1966). Thus, the fixative perfusion rate was adapted to animal body weight and a peripheral vessel was sectioned.

For foetal kidney studies the abdomen of pregnant females was opened and the uterus exposed. The fixative (glutaraldehyde 1 % plus paraformaldehyde 0·8% in 0·12 M cacodylate or phosphate buffer with an admixture of 0·25% sodium chloride, pH 7· 3) was introduced through a fine hypodermic needle into the vitelline vein at a flow rate of 10 μl/min (15-to 18-day-old foetuses) or 20 μl /min (19-and 21-day-old foetuses) for 20 min.

In postnatal stages the fixative was back perfused into the abdominal aorta proximal to its distal bifurcation, just after clamping the aorta above the renal pedicles, at a flow rate of 20μl /min (newborn and 3-day-old rats), 50μl /min (25-day-old rats) or 100 μl /min (adults) for 20 min. The kidneys were then quickly removed, immediately immersed in the fixative and cut perpendicularly to the renal capsule into pieces of 2– 3 mm³ which were transferred into vials containing fresh fixative at room temperature for 3 h. After a 10 min rinse in 0· 12 M cacodylate or phosphate buffer containing 0·25% sodium chloride, pH 7· 3, tissues were stored overnight at 4 °C in the same solution and subsequently post-fixed with 2% OsO₄ in 0· 1 M cacodylate or phosphate buffer, pH 7· 3 at 4 °C for 1 h. After washing successively with buffer (15 min) and distilled water (three washes of 10 min each), tissues were dehydrated in graded series of ethanol and embedded in Epon-Araldite (Mollenhauer & Totten, 1971). The blocks were cut with glass knives on a Porter-Blum MT2 ultramicrotome. Thick sections (1· 5 μ m) perpendicular to the kidney capsule were stained in a mixture 1/1 (v/v) of 1% methylene blue in saturated sodium borate and 1 % of Azure blue in water, and then examined by light microscopy in order to localize the most differentiated nephron fields. In these selected fields, ultra-thin sections were cut, sequentially stained in a mixture 1/1 (v/v) of a saturated aqueous solution of uranyl acetate and pure acetone for 20 min and in lead citrate (Reynolds, 1963) for 6 min. They were examined in a Hitachi HS-8 electron microscope at 50 kV.

Urine samples were collected by transbladder aspiration. Proteins were separated by electrophoresis in 4– 26% polyacrylamide gel gradient slabs (electrode buffer: Tris-borate-EDTA, pH 8· 35, according to Kitchin (1965), at 220 V and 10 °C for 24 h). The slabs were stained with Coomassie blue (0· 1 % solution in methanol-acetic acid-water: 10/1/10, v/v).

The protein tracer (horseradish peroxidase type II, Sigma Chemical Company′, molecular weight: 40000) was dissolved in physiological saline and perfused in a small volume to prevent hemodynamical changes which are known to affect the glomerular transport of macromolecules (Ryan & Karnovsky, 1976). Thus, horseradish peroxidase, 100– 200 μ g per g of body weight was infused over 1 min into the vitelline vein of 17-to 21-day-old foetuses in volumes of 5– 10 μl according to their age. Animals were fixed by perfusion 1, 2, 4, 6, 8, 10, 15 or 20 min after horseradish peroxidase injection (three or four animals for each interval) as described above. After additional fixation by immersion in the glutaraldehyde-paraformaldehyde solution, tissues were rinsed at once in 0· 12 M cacodylate or phosphate buffer plus 0· 25 % sodium chloride, pH 7· 3, overnight and then in 0· 05 M Tris-HCl buffer, pH 7· 6, for 30 min. Peroxidase activity was revealed according to Graham & Karnovsky′s method (1966a) on small pieces excised with a razor blade (when we attempted to use frozen 40 μ m slices, the tissue was torn during successive manipulations since foetal kidney is quite delicate). The kidneys from two animals injected with saline solution were studied as controls. After incubation in test medium, tissues were rinsed, post-fixed in osmium tetroxide, dehydrated and embedded in Epon-Araldite. The sections were examined without additional staining.

Only the most differentiated, that is the deepest nephrons, will be described at every stage of foetal development.

The first structures identifiable as nephrons appear. They are still scattered in a tissue composed of sparse cells and are seen as discrete masses in the immediate vicinity of the end of a collecting tubule.

These nephrogenic buds develop a central lumen rapidly and become renal vesicles. The most differentiated nephrons are S-shaped bodies resulting from the elongation and curving of renal vesicles. These S-shaped bodies are completely surrounded by a very thin basement lamina. At this developmental stage, intercellular spaces are locally irregular in width and all cells exhibit almost the same ultrastructure. Each nucleus contains several large nucleoli.

Ribosomes and polysomes are numerous but only a few long channels of rough endoplasmic reticulum are seen. Golgi apparatus presents very short saccules. Mitochondria are small and randomly dispersed. Microtubules, parallel to the plasma membrane, are restricted to the basal half of the cell, on both sides of the nucleus.

The lower limb of the S-shaped body increases in width and forms a cup, the centre of which is invaded by a capillary loop. This cup deepens and constitutes a double-walled hemisphere. The cells of the outer wall soon begin to flatten and will become the Bowman’s capsule; the cells of the inner wall are the future podocytes. The two walls are separated by a narrow space. The early podocytes have a cuboidal shape and their lateral cell membranes are closely apposed. No foot process can be seen. The capillary wall is thick and without fenestra’, endothelial cells have irregular surfaces with thin évaginations inside the capillary lumen and, facing the podocytes, flat cytoplasmic expansions tangential to the capillary wall section. The space between the epithelium and the endothelium is irregular in width (from 0· 15 to 0· 45 μ m) and occupied by a thin basement lamina located next to the podocytes (Fig. 1).

The middle limb of the S-shaped body will give rise to the proximal tubule which is always seen close to the glomerulus in sections. The tubular lumen is very narrow and the lining cells are connected by a long subapical intermediate junction (Zonula adherens). The distribution of cellular organelles is the same as previously (Fig. 2).

The S-shaped body is connected to the collecting tubule. The podocytes appear still as cuboidal cells and are joined by several junctions near their capillary side. Elsewhere, they are separated from each other by wide intercellular spaces. A few broad and short processes begin to develop on the cell surface adjacent to the capillary; they probably represent the first-order podocyte branches. Endothelial cells have a thick and non-fenestrated cytoplasm. As before, a thin basement lamina from which filamentous material extends up to the endothelial cells, is seen close to the podocyte membrane (Figs. 3, 4).

Some of the proximal tubule sections are located next to the glomerulus while others are apart, suggesting that these tubules are longer than in the preceding stage. Adjacent cells show several kinds of junctions: one short tight junction near the tubular lumen, one junction just beneath, and several gap junctions up to the base of the cells. A few short microvilli first appear at this stage. In the underlying cytoplasm there are tubular invaginations, small vesicles and, towards the base, a few lysosomes and autophagic vacuoles. In the lower part of the cell, scanty peroxisomes and lipid droplets can be seen. The other cytoplasmic organelles have the same structure and distribution as before (Fig. 5).

The podocytes have an irregular shape and come apart from each other except near the “basement membrane where they are fastened together by a few junctions. The urinary space is about 4 μ m wide. Broad foot processes take shape facing the endothelial cells, the slits between them being bridged by a diaphragm ; elsewhere, cell membranes exhibit scarce and short cytoplasmic extensions. The capillary wall is generally thick but occasionally thin and even fenestrated, pores being obstructed by electron-dense material. Besides the podocyte basement lamina, a thin discontinuous lamina is seen next to the endothelial cells. These two laminae are joined by fine filaments (Figs. 6,7). In the proximal tubule, the microvilli are more numerous and longer than in the previous stage. The cell membranes present a few basal and lateral infoldings. Most of the apical endocytic vesicles are small (0· 4 μ m in diameter) and a few middle-sized (between 0· 4 and 0· 8 μ m in diameter), both bearing a thick internal coat (Fig. 8). Mitochondria remain small and scattered. Golgi apparatus consists of short saccules and is located on both sides of the nucleus in a direction parallel to the lateral plasma membrane.

The foot processes are longer than in the previous stage. In the endothelial cells a few pores obstructed by a fibrillar material are observed. The basement lamina of these cells is continuous and separated from that of the epithelial cells by a thin filamentous zone of low electron density (Fig. 9, 10).

The proximal tubule microvilli increase in length. There are only a few plasma membrane invaginations on the basal and lateral sides of the cells. The apical-coated vesicles are numerous and uncoated or thinly coated large vesicles (1 μ m in diameter) appear just beneath. The microtubules, either alone or in bundles, are located not only alongside the nucleus but also in the apical cytoplasm of the cells, especially between the endocytic vesicles. The mitochondria, similar in size as in the preceding developmental stage, are found alongside channels of rough endoplasmic reticulum (Fig. 11).

The podocytes present many long and thin foot processes and are tightly packed so that, in sections, their monolayer disposition is no longer obvious. They enlarge and tend to fill the urinary space which is consequently reduced to interpodocyte gaps, as in the mature glomerulus. The capillary loops are twisted and intermixed with epithelial and mesangial cells. A very thin layer of endothelium extends around the capillary and shows many open pores. The epithelial and endothelial cells draw close to one another and then share a common basement membrane resulting probably from the coalescence of previous thin laminae. On both sides of this preliminary lamina densa, one sees a loose network which will become the laminae rarae (Fig. 12).

The proximal tubule cells present even longer microvilli. The main feature of this developmental stage is the presence of profuse large endocytic vesicles sometimes seen close by lysosome-like bodies. Golgi apparatus contains saccules longer than in the previous stage (about 2μ m). The mitochondria enlarge, come together at the basal half of the cells and show a tendency to be oriented perpendicularly to the tubule axis. They are bordered by channels of rough endoplasmic reticulum (Fig. 13).

The former features of glomerulus differentiation are strongly marked. This stage is more particularly characterized by a thickening of the glomerular basement membrane to about 0· 1 μ m in width (Fig. 14)

The proximal tubule cells have extensively interdigitating processes which contain long mitochondria arranged perpendicular to the tubule axis, close to the plasma membrane. The endocytic vesicles are very numerous in the apical part of the cells, the large ones being almost contiguous to each other. Some large vesicles are seen apparently in the process of fusing with lysosomes (Fig. 15).

The structure of the most differentiated nephrons is almost the same as in the last foetal stage, except that the number and the size of the large endocytic vesicles are increased.

The process of intermingling of the epithelial and endothelial cells becomes more accentuated than previously and the glomerular basement membrane is as thick as in adult stage (0· 15 μ m).

In the proximal tubule, there are a few small vesicles, while the large ones remain numerous, most of them being probably engaged in fusion with lysosomes. Voluminous droplets are observed at the base of the cells (Fig. 16).

The nephrons have the same structure as in the adult. In particular, the proximal tubules show many long and slender microvilli and only a few endocytic vesicles. Basal and lateral infoldings are very deep, setting up high septa between which are seen very long mitochondria fringed with rough endoplasmic reticulum channels.

Foetal electrophoregrams show the presence of most of the plasma proteins, including high-molecular-weight ones, such as α -2-macroglobulin (molecular weight: 800000). In the urine of young animals (3-day-old) the same components are found except for the proteins of molecular weight above 150000– 200000 which are absent. In the urine of adults only a few traces of light proteins are present (Fig. 17).

Before day 18 of gestation, peroxidase reaction product cannot be detected either in the glomerulus or in the proximal tubule. At day 18, enzyme activity is found after 4 min of intravenous injection of peroxidase in the glomerular basement membrane, in the urinary space (Fig. 18), on the proximal tubule microvilli, in the tubular invaginations and in the small apical vesicles (Fig. 19). At day 19 of gestation, peroxidase filtration occurs as early as 2 min after perfusion is stopped and reaction product is observed in the apical tubules of the proximal tubule cells. Two minutes later all the apical vesicles contain reaction product at their periphery. At days 20 and 21 filtration is still faster, starting from 1 min following peroxidase injection (Fig. 20). After 2 min, all the small apical vesicles contain peroxidase and the reaction product is usually located on the inside of the vesicle membrane (Fig. 21); on the contrary, large vesicles which fuse with lysosomes are filled with scattered precipitates which soon become less and less contrasted (Figs. 22, 23). In addition, from day 18 onwards, some peroxidase activity is observed in intercellular spaces, but this is probably a result of enzyme diffusion from peritubular capillaries.

Foetal urine analysis shows the presence of many plasma proteins as compared with the urine of adult animals. This proteinuria seems to be largely due to immaturity of the glomerular filtration barrier since even high-molecular-weight proteins, which do not permeate the glomerular filter in the differentiated nephron (Graham & Karnovsky, 1966b), are found in foetal urine. Two kinds of observations are in agreement with this assumption. Firstly, at the start of glomerular filtration, indicated by the first detection of horseradish peroxidase in the urinary space (namely at day 18), the glomerular basement membrane is merely composed of a thin lamina probably much more permeable than the thick three-layered lamina of the mature glomerulus. Although we are unaware of the composition of the glomerular filtrate at the different stages of nephron differentiation, it is likely that selective permeability appears gradually with successive biochemical deposits onto the glomerular barrier. Secondly, the endocytic apparatus, which comprises the apical vesicles and the secondary lysosomes resulting from coalescence between the large endocytic vesicles and the primary lysosomes, is clearly more profuse at foetal stages than in the mature tubule. This may be due to a relatively high concentration of plasma proteins in the glomerular filtrate. Indeed, it has been suggested that the number of endocytic vesicles increases either in proximal tubule (Bergelin & Karlsson, 1975; Larsson & Maunsbach, 1975) or in other cells (Cohn & Fedorko, 1969) when they are exposed to increasing concentrations of macromolecules.

Foetal proteinuria may also be related to immaturity of the tubular reabsorption system. In the differentiated proximal tubule, nearly all the filtered proteins are picked up into endocytic vesicles via the apical tubular invaginations and then digested into secondary lysosomes (Larsson & Maunsbach, 1975). As indicated by tracer studies, the whole endocytic process occurs within a few minutes either in the mature nephron or at the late stages of its differentiation (days 20 and 21 of gestation). On the contrary, in the early stages (days 18 and 19 of gestation) the formation of secondary lysosomes takes place only 1 or 2 days after the onset of endocytosis, though the primary lysosomes are present from the first steps of tubule development. The congestion of apical vesicles, possibly consequent on the lack of formation of secondary lysosomes, may be the cause of a slower endocytosis; this, in addition to the abundance of plasma proteins in the glomerular filtrate, may account for foetal proteinuria. However, that cannot explain the presence of large protein molecules in foetal urine since protein reabsorption in the proximal tubule is a non-specific phenomenon : therefore, any qualitative changes in the composition of urine, i.e. the presence or the absence of large protein molecules, is likely to be a consequence of glomerular permeability.

The gradual differentiation of the protein reabsorption mechanisms is perhaps in close relation to the cellular distribution of microtubules. So, it is noteworthy that the apical ordering of microtubules takes place just before the first appearance of secondary lysosomes. Previously, they were located along the lateral cell membranes to the exclusion of apical cytoplasm. Since microtubules are involved in intracellular movement (Silverblatt, Tyson & Bulger, 1974) it is possible that the delayed appearance of secondary lysosomes in immature proximal tubules results from the lack of apical microtubules as endocytosis commences.

In other respects, as shown by urine electrophoresis, proteinuria is more selective in the newborn than in the foetus, in spite of the presence of immature nephrons for several days after birth. It is possible that the difference between prenatal and postnatal selective permeability is at least partially due to an earlier differentiation of the glomerular basement membrane in postnatal than in foetal developing nephrons. Thus, in the postnatal kidney, Kazimierczak (1971) and Larsson & Maunsbach (1975) have reported that the epithelial and endothelial basement lamina occasionally merge as early as the S stage and that a three-layered basement membrane is observed when the endothelial cells have only a few pores, whereas, in the foetal kidney, we observe a thin epithelial lamina at the S-shaped-body stage and a typical basement membrane only when the capillary wall is largely fenestrated.

In conclusion, the present study shows that the transient proteinuria observed during foetal life is chiefly due to the immaturity of the glomerular barrier.

This investigation was supported by Institut National de la Santé et de la Recherche Médicale (ATP 62-78-94).