differentiation of glomerular filter and tubular ... · and tubular reabsorption apparatus during...

/ . Embryol. exp. Morph. Vol. 58, pp. 157-175,1980 \ 57Printed in Great Britain © Company of Biologists Limited 1980

Differentiation of glomerular filterand tubular reabsorption apparatus during foetal

development of the rat kidney

By JEAN SCHAEVERBEKE AND MADELEINE CHEIGNON1

From the Laboratoire de Biologie Cellulaire,Universite Paris VII

SUMMARYDifferentiation of the glomerulus and the proximal tubule was studied in the rat foetus,

especially with regard to the development of the protein filtration-reabsorption apparatus.Filtration starts several days before full differentiation of the glomerulus, when the glomerularbasement membrane consists of a thin lamina alongside the podocyte membrane. Endocytosisis functional from this time, but fusion between endocytic vesicles and lysosome-like bodiesoccurs 2 days later. Foetal urine electrophoresis shows the presence of many proteins,including high molecular weight ones, this proteinuria seeming chiefly due to the immaturityof the glomerular barrier.

INTRODUCTION

Differentiation of the nephron and particularly of the glomerulus has beeninvestigated 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 thecorrelations between the structural development of the protein retention system(glomerular filtration barrier and tubular reabsorption apparatus) and theonset of filtration and selective permeability properties.

Development of the metanephros begins during foetal life but is onlycompleted after birth. All the nephrons do not develop at the same time. Thefirst nephrogenic masses appear under the kidney capsule and are progressivelydifferentiated and connected to the collecting tubules; new nephron generationsare 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, mostnephrons appear as mature formations but in certain species, particularly inthe rat, some nephrogenic masses can still be seen, their differentiation beingachieved several days or weeks following birth (Kasimierczak, 1971; Larsson1975; Kasimierczak, 1976).

1 Authors' address; Laboratoire de Biologie Cellulaire, Universite Paris VII, Tour 23-33ler etage, 2 place Jussieu, 75221 Paris Cedex 05, France.

11-2

158 J. SCHAEVERBEKE AND M. CHEIGNON

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

MATERIALS AND METHODS

Animals

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

Ultrastructural differentiation

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

For foetal kidney studies the abdomen of pregnant females was openedand the uterus exposed. The fixative (glutaraldehyde 1 % plus paraformaldehyde0-8% 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 needleinto the vitelline vein at a flow rate of 10/4/min (15- to 18-day-old foetuses)or 20 [A I mm (19- and 21-day-old foetuses) for 20 min.

In postnatal stages the fixative was back perfused into the abdominal aortaproximal to its distal bifurcation, just after clamping the aorta above the renalpedicles, at a flow rate of 20 /d/min (newborn and 3-day-old rats), 50 /tl/min(25-day-old rats) or 100 /tl/min (adults) for 20 min. The kidneys were thenquickly removed, immediately immersed in the fixative and cut perpendicularlyto the renal capsule into pieces of 2-3 mm3 which were transferred into vialscontaining fresh fixative at room temperature for 3 h. After a 10 min rinse in0-12 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 sub-sequently 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) anddistilled water (three washes of 10 min each), tissues were dehydrated in gradedseries of ethanol and embedded in Epon-Araldite (Mollenhauer & Totten,1971). The blocks were cut with glass knives on a Porter-Blum MT2 ultra-microtome. Thick sections (1-5 /on) perpendicular to the kidney capsule werestained in a mixture 1/1 (v/v) of 1 % methylene blue in saturated sodium

Differentiation in foetal rat kidney 159borate and 1 % of Azure blue in water, and then examined by light microscopyin order to localize the most differentiated nephron fields. In these selectedfields, 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 minand in lead citrate (Reynolds, 1963) for 6 min. They were examined in aHitachi HS-8 electron microscope at 50 kV.

Qualitative analysis of urinary proteins

Urine samples were collected by transbladder aspiration. Proteins wereseparated by electrophoresis in 4-26% polyacrylamide gel gradient slabs(electrode buffer: Tris-borate-EDTA, pH 8-35, according to Kitchin (1965), at220 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).

Experiments with horseradish peroxidase

The protein tracer (horseradish peroxidase type II, Sigma Chemical Company,molecular weight: 40000) was dissolved in physiological saline and perfusedin a small volume to prevent hemodynamical changes which are known toaffect the glomerular transport of macromolecules (Ryan & Karnovsky, 1976).Thus, horseradish peroxidase, 100-200 /*g per g of body weight was infusedover 1 min into the vitelline vein of 17- to 21-day-old foetuses in volumes of5-10/d 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 animalsfor each interval) as described above. After additional fixation by immersionin the glutaraldehyde-paraformaldehyde solution, tissues were rinsed at oncein 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. Peroxidaseactivity was revealed according to Graham & Karnovsky's method (1966a) onsmall pieces excised with a razor blade (when we attempted to use frozen40 /tin slices, the tissue was torn during successive manipulations since foetalkidney is quite delicate). The kidneys from two animals injected with salinesolution were studied as controls. After incubation in test medium, tissueswere rinsed, post-fixed in osmium tetroxide, dehydrated and embedded inEpon-Araldite. The sections were examined without additional staining.

RESULTS

Ultrastructural differentiation

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

Day 15 of gestation. The first structures identifiable as nephrons appear.They are still scattered in a tissue composed of sparse cells and are seen asdiscrete masses in the immediate vicinity of the end of a collecting tubule.

J. SCHAEVERBEKE AND M. CHEIGNON

Fig. 1. Glomerulus of 16-day-old foetus. The epithelial cell (Ep) has no footprocess. The endothelial cell {En) is thick, without fenestra. The space betweenthe epithelium and the endothelium is occupied by a thin basement lamina (B)located next to the podocytes and by a sparse material alongside the endothelialcells, x 28000.Fig. 2. Proximal tubule of 16-day-old foetus. The cells are high and connected byan intermediate junction. The tubular lumen (TL) is narrow. Very short microvillican be seen, x 6000.

Differentiation in foetal rat kidney 161

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Fig. 3. Glomerulus of 17-day-old foetus. The epithelial cell (Ep) has no foot processand presents some cytoplasmic invaginations near the urinary space (US). Theendothelial cell (En) is thick, without fenestra. B, Future glomerular basementlamina, x 6500.

Fig. 4. Glomerulus of 17-day-old foetus. The epithelial cell (Ep) has only a fewshallow infoldings. The space between podocytes end endothelium is occupied bya thin basement lamina (arrow) from which filamentous material extends up to theendothelial cell (En), x 27000.Fig. 5. Proximal tubule of 17-day-old foetus. Note the presence of short microvilli(Mi) and apical tubular invaginations (at). Polysomes, lysosomes (Ly) and smallmitochondria are dispersed in the cytoplasm. The intercellular space is narrow,without infolding, x 12000.

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


1*

IS*Fig. 6. Glomerulus of 18-day-old foetus. The epithelial cells have an irregularshape. In the endothelial cell (En), there are a few fenestrae irregularly arranged.US, urinary space; B, glomerular basement lamina, x 8000.Fig. 7. Glomerulus of 18-day-old foetus. The glomerular basement lamina iscomposed of two layers: a continuous lamina close to the podocytes (large arrow)and a discontinuous one next to the endothelium (En). The epithelial cell (Ep)presents broad foot processes (Fp). Slit diaphragms can be seen bridging thenarrow gap between neighbouring foot processes (small arrow), x 36000.Fig. 8. Proximal tubule of 18-day-old foetus. Mi, Microvilli; at, apical tubular in-vaginations; sav, small apical vesicles, x 30000.

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Differentiation in foetal rat kidney 163Ribosomes and polysomes are numerous but only a few long channels ofrough endoplasmic reticulum are seen. Golgi apparatus presents very shortsaccules. 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.

Day 16 of gestation. The lower limb of the S-shaped body increases in widthand forms a cup, the centre of which is invaded by a capillary loop. This cupdeepens and constitutes a double-walled hemisphere. The cells of the outerwall soon begin to flatten and will become the Bowman's capsule; the cellsof the inner wall are the future podocytes. The two walls are separated by anarrow space. The early podocytes have a cuboidal shape and their lateralcell membranes are closely apposed. No foot process can be seen. The capillarywall is thick and without fenestra; endothelial cells have irregular surfaceswith thin evaginations inside the capillary lumen and, facing the podocytes,flat cytoplasmic expansions tangential to the capillary wall section. The spacebetween the epithelium and the endothelium is irregular in width (from 0-15to 0-45 /.tin) and occupied by a thin basement lamina located next to thepodocytes (Fig. 1).

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

Day 17 of gestation. The S-shaped body is connected to the collecting tubule.The podocytes appear still as cuboidal cells and are joined by several junctionsnear their capillary side. Elsewhere, they are separated from each other bywide intercellular spaces. A few broad and short processes begin to developon the cell surface adjacent to the capillary; they probably represent thefirst-order podocyte branches. Endothelial cells have a thick and non-fenestratedcytoplasm. As before, a thin basement lamina from which filamentous materialextends 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 glomeruluswhile others are apart, suggesting that these tubules are longer than in thepreceding stage. Adjacent cells show several kinds of junctions: one shorttight junction near the tubular lumen, one junction just beneath, and severalgap junctions up to the base of the cells. A few short microvilli first appearat this stage. In the underlying cytoplasm there are tubular invaginations, smallvesicles and, towards the base, a few lysosomes and autophagic vacuoles. Inthe lower part of the cell, scanty peroxisomes and lipid droplets can be seen.The other cytoplasmic organelles have the same structure and distribution asbefore (Fig. 5).

Day 18 of gestation. The podocytes have an irregular shape and come apart

Differentiation in foetal rat kidney 165from each other except near the [basement membrane where they are fastenedtogether by a few junctions. The urinary space is about 4 fim wide. Broad footprocesses take shape facing the endothelial cells, the slits between them beingbridged by a diaphragm; elsewhere, cell membranes exhibit scarce and short cyto-plasmic extensions. The capillary wall is generally thick but occasionally thinand even fenestrated, pores being obstructed by electron-dense material. Besidesthe podocyte basement lamina, a thin discontinuous lamina is seen next tothe endothelial cells. These two laminae are joined by fine filaments (Figs. 6,7).

In the proximal tubule, the microvilli are more numerous and longer thanin the previous stage. The cell membranes present a few basal and lateralinfoldings. Most of the apical endocytic vesicles are small (0-4 /im in diameter)and a few middle-sized (between 0-4 and 0-8 /im in diameter), both bearing athick internal coat (Fig. 8). Mitochondria remain small and scattered. Golgiapparatus consists of short saccules and is located on both sides of the nucleusin a direction parallel to the lateral plasma membrane.

Day 19 of gestation. The foot processes are longer than in the previous stage.In the endothelial cells a few pores obstructed by a fibrillar material areobserved. The basement lamina of these cells is continuous and separated fromthat 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 fewplasma membrane invaginations on the basal and lateral sides of the cells. Theapical-coated vesicles are numerous and uncoated or thinly coated largevesicles (1 fim in diameter) appear just beneath. The microtubules, either aloneor in bundles, are located not only alongside the nucleus but also in the apicalcytoplasm of the cells, especially between the endocytic vesicles. The mito-chondria, similar in size as in the preceding developmental stage, are foundalongside channels of rough endoplasmic reticulum (Fig. 11).

Day 20 of gestation. The podocytes present many long and thin foot processesand are tightly packed so that, in sections, their monolayer disposition is nolonger obvious. They enlarge and tend to fill the urinary space which is con-sequently reduced to interpodocyte gaps, as in the mature glomerulus. The

FIGURES 9-11

Fig. 9. Glomerulus of 19-day-old foetus. The glomerular basement membrane iscomposed of two continuous layers (arrows) sparsely joined by a filamentousmaterial, x 37000.Fig. 10. Glomerulus of 19-day-old foetus. Ep, Epithelial cell; Fp, foot process;En, endothelial cell; B, glomerular basement membrane; US, urinary space,x 23 000.Fig. 11. Proximal tubule of 19-day-old foetus. At the base of the microvilli, endo-cytic vesicles in formation can be seen (arrows). Membrane of the small apicalvesicles (sav) is covered by a thick internal coat, x 34000.


Fig. 12. Glomerulus of 20-day-old foetus. The glomerular basement membrane(B) is composed of a lamina densa bordered on both sides by two laminae rarae.The endothelium (En) is fenestrated CO- Slit diaphragm, arrow; Ep, epithelial cell;Fp, foot process, x 30000.Fig. 13. Proximal tubule of 20-day-old foetus, sav, small apical vesicles; lav, largeapical vesicle; Ly, lysosome; bi, basal infolding, x 11000.

capillary loops are twisted and intermixed with epithelial and mesangial cells.A very thin layer of endothelium extends around the capillary and shows manyopen pores. The epithelial and endothelial cells draw close to one another andthen share a common basement membrane resulting probably from the co-alescence of previous thin laminae. On both sides of this preliminary laminadensa, one sees a loose network which will become the laminae rarae (Fig. 12).


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Fig. 14. Glomerulus of 21-day-old foetus. The structure is the same as in theprevious stage except a thickening of the lamina densa. Ep, Epithelial cell; Fp, footprocess; slit diaphragm, arrow; B, glomerular basement membrane; En, endo-thelialcell;/, fenestra. x 30000.Fig. 15. Proximal tubule of 21-day-old foetus. The small (sav) and large (lav) apicalendocytic vesicles are numerous. Mi, microvilli; Ly, lysosome. x 21000.

The proximal tubule cells present even longer microvilli. The main featureof this developmental stage is the presence of profuse large endocytic vesiclessometimes seen close by lysosome-like bodies. Golgi apparatus containssaccules longer than in the previous stage (about 2/*m). The mitochondriaenlarge, come together at the basal half of the cells and show a tendency tobe oriented perpendicularly to the tubule axis. They are bordered by channelsof rough endoplasmic reticulum (Fig. 13).

Day 21 of gestation. The former features of glomerulus differentiation are


Fig. 16. Proximal tubule of 3-day-old animal. The small apical vesicles (sav) areless numerous than previously. The large vesicles (lav) are seen close to lysosome(Ly), these two organelles being likely in process of fusion. Mi, microvilli. x 13000.

Differentiation in foetal rat kidney

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169

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17

Fig. 17. Electrophoresis of rat urine in a 4-26% polyacrylamide gel gradient.A, 21-day-old foetus; B, 3-day-old animal; C, adult. 1, prealbumins; 2, albumin;3, a-foetoprotein; 4, a2-macroglobulin.

strongly marked. This stage is more particularly characterized by a thickeningof the glomerular basement membrane to about 0-1 /tm in width (Fig. 14)

The proximal tubule cells have extensively interdigitating processes whichcontain long mitochondria arranged perpendicular to the tubule axis, closeto the plasma membrane. The endocytic vesicles are very numerous in theapical 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).

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

3-day-old animals. The process of intermingling of the epithelial andendothelial cells becomes more accentuated than previously and the glomerularbasement membrane is as thick as in adult stage (0-15 /on).

In the proximal tubule, there are a few small vesicles, while the large onesremain numerous, most of them being probably engaged in fusion with

Differentiation in foetal rat kidney 111lysosomes. Voluminous droplets are observed at the base of the cells (Fig. 16).

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

Development of protein filtration-reabsorption properties

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

Location of horseradish peroxidase after intravenous injection. Before day 18of gestation, peroxidase reaction product cannot be detected either in theglomerulus or in the proximal tubule. At day 18, enzyme activity is foundafter 4 min of intravenous injection of peroxidase in the glomerular basementmembrane, in the urinary space (Fig. 18), on the proximal tubule microvilli, inthe tubular invaginations and in the small apical vesicles (Fig. 19). At day 19of gestation, peroxidase filtration occurs as early as 2 min after perfusion isstopped and reaction product is observed in the apical tubules of the proximaltubule cells. Two minutes later all the apical vesicles contain reaction productat their periphery. At days 20 and 21 filtration is still faster, starting from 1 minfollowing peroxidase injection (Fig. 20). After 2 min, all the small apicalvesicles contain peroxidase and the reaction product is usually located on theinside of the vesicle membrane (Fig. 21); on the contrary, large vesicles which

FIGURES 18-21

Fig. 18. Glomerulus of 18-day-old foetus fixed 4 min after horseradish peroxidaseinjection. The reaction product (arrow) is seen in the glomerular basement mem-brane (B) between epithelial (Ep) and endothelial (En) cells, especially alongsidethe podocyte. Unstained, x 34000.Fig. 19. Proximal tubule of 18-day-old foetus fixed 4 min after horseradish peroxidaseinjection. The reaction product is present on the brush border, in the apical tubularinvaginations and in the small vesicles farrows). Unstained, x 16000.Fig. 20. Glomerulus of 21-day-old foetus fixed 1 min after horseradish peroxidaseinjection. The reaction product (arrows) is present in the capillary lumen, in theglomerular basement membrane (B), on the podocyte membrane and in the urinaryspace (US). Ep, Epithelial cell; Fp, foot process; En, endothelial cell. Unstained.x 30000.Fig. 21. Proximal tubule of 21 -day-old foetus fixed 2 min after horseradish peroxidaseinjection. The tracer (arrows), present on the brush border membrane, is pickedup into small vesicles (sav). Unstained, x 31000.12 EMB 58


Fig. 22. Apical cytoplasm of a proximal tubule cell of 21-day-old foetus fixed4 min after horseradish peroxidase injection. The tracer (arrow) is present on themicrovilli, in the apical tubular invaginations and in the large apical vesicles (lav)in fusing process with a lysosome (Ly); Unstained, x 18000.Fig. 23. Proximal tubule of 21-day-old foetus fixed 10 minutes after horseradishperoxidase injection. The microvilli (Mi) and the small apical vesicles (sav) lackingreaction product. The large apical vesicles (lav) contain the reaction product(arrow). Ly, lysosome. Unstained, x 13000.

fuse with lysosomes are filled with scattered precipitates which soon becomeless and less contrasted (Figs. 22, 23). In addition, from day 18 onwards, someperoxidase activity is observed in intercellular spaces, but this is probablya result of enzyme diffusion from peritubular capillaries.

DISCUSSIONFoetal urine analysis shows the presence of many plasma proteins as compared

with the urine of adult animals. This proteinuria seems to be largely due toimmaturity of the glomerular filtration barrier since even high-molecular-weightproteins, which do not permeate the glomerular filter in the differentiatednephron (Graham & Karnovsky, 19666), are found in foetal urine. Two kindsof observations are in agreement with this assumption. Firstly, at the start ofglomerular filtration, indicated by the first detection of horseradish peroxidase


in the urinary space (namely at day 18), the glomerular basement membraneis merely composed of a thin lamina probably much more permeable than thethick three-layered lamina of the mature glomerulus. Although we are unawareof the composition of the glomerular filtrate at the different stages of nephrondifferentiation, it is likely that selective permeability appears gradually withsuccessive biochemical deposits onto the glomerular barrier. Secondly, theendocytic apparatus, which comprises the apical vesicles and the secondarylysosomes resulting from coalescence between the large endocytic vesicles andthe primary lysosomes, is clearly more profuse at foetal stages than in themature tubule. This may be due to a relatively high concentration of plasmaproteins in the glomerular filtrate. Indeed, it has been suggested that thenumber 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 macro-molecules.

Foetal proteinuria may also be related to immaturity of the tubular reab-sorption system. In the differentiated proximal tubule, nearly all the filteredproteins are picked up into endocytic vesicles via the apical tubular invagi-nations and then digested into secondary lysosomes (Larsson & Maunsbach,1975). As indicated by tracer studies, the whole endocytic process occurswithin a few minutes either in the mature nephron or at the late stages of itsdifferentiation (days 20 and 21 of gestation). On the contrary, in the earlystages (days 18 and 19 of gestation) the formation of secondary lysosomestakes place only 1 or 2 days after the onset of endocytosis, though the primarylysosomes are present from the first steps of tubule development. The con-gestion of apical vesicles, possibly consequent on the lack of formation ofsecondary lysosomes, may be the cause of a slower endocytosis; this, in additionto the abundance of plasma proteins in the glomerular filtrate, may accountfor foetal proteinuria. However, that cannot explain the presence of largeprotein molecules in foetal urine since protein reabsorption in the proximaltubule is a non-specific phenomenon: therefore, any qualitative changes in thecomposition 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 isperhaps in close relation to the cellular distribution of microtubules. So, it isnoteworthy that the apical ordering of microtubules takes place just beforethe first appearance of secondary lysosomes. Previously, they were locatedalong the lateral cell membranes to the exclusion of apical cytoplasm. Sincemicrotubules are involved in intracellular movement (Silverblatt, Tyson &Bulger, 1974) it is possible that the delayed appearance of secondary lysosomesin immature proximal tubules results from the lack of apical microtubules asendocytosis 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 immaturenephrons for several days after birth. It is possible that the difference betweenprenatal and postnatal selective permeability is at least partially due to anearlier differentiation of the glomerular basement membrane in postnatal thanin foetal developing nephrons. Thus, in the postnatal kidney, Kazimierczak(1971) and Larsson & Maunsbach (1975) have reported that the epithelialand endothelial basement lamina occasionally merge as early as the S stageand that a three-layered basement membrane is observed when the endothelialcells have only a few pores, whereas, in the foetal kidney, we observe a thinepithelial lamina at the S-shaped-body stage and a typical basement membraneonly when the capillary wall is largely fenestrated.

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

This investigation was supported by Institut National de la Sante et de la RechercheMedicale (ATP 62-78-94).

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MIYOSHI, M., FUJITA, T. & TOKUNAGA, J. (1971). The differentiation of renal podocytes: acombined scanning and electron microscope study in rats. Archs Histol. Jap. 33, 161—178.

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SILVERBLATT, F. J., TYSON, G. E. & BULGER, R. E. (1974). Effects of vinblastine on thephagolysosome system of proximal tubule cells of rat kidney: administration of horse-radish peroxidase. Lab. Invest. 31, 170-180.

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(Received 30 October 1979, revised 9 February 1980)

differentiation of glomerular filter and tubular ... · and tubular reabsorption apparatus during...

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