Abstract We have investigated the temporal maturationof the rat kidney during the postnatal developmental pe-riod. As a result, we observed the following: an activeprocess of cortical cell proliferation and differentiationoccurs as late as day 20. The medulla is the most imma-ture zone at birth and displays the greatest morphologi-cal changes during this period. At birth, no distinctionexists between inner and outer medulla, and the outerand inner strip of the outer medulla can be distinguishedas late as day 30. Remodeling of the ECM surroundingcollecting ducts occurs in the medulla twice, stopping atday 11 and it occurs in the papilla three times, stoppingat day 20. The increase of kidney size is temporally dif-ferent for each kidney zone. The cortex and the papillaacquire the morphological appearance of the adult kid-ney before the medulla does. Consequently, the medullaremains at the highest degree of immaturation among thekidney zones for a relatively long postnatal period.

Keywords Kidney · Postnatal development · Proliferation · Lectin binding

Introduction

The embryonic development of the kidney has been wellcharacterized (Saxen 1987, Clark and Bertram 1999).The development of nephrons in the permanent kidney(metanephrogenesis) starts at about 5 weeks of gestation

in humans and is completed in utero before gestationalweek 36 (Nigam et al. 1996). In mice and rats, nephro-genesis begins on embryonic days 11 and 12, respective-ly, and is considered completed at about 7–10 days afterbirth (Speller and Moffat 1977, Nigam et al. 1996). Atbirth, the mammalian kidney is very immature and allstages of renal glomerular and tubular development canbe visualized in a histological section of the newborn ratkidney (Mugrauer and Ekblom 1991, Dodge 1997).

The adult kidney is a highly stratificated organ, bothmorphologically and functionally. The function of thekidney is to maintain the salt, water and acid-base bal-ance of the body and to secrete waste products in theform of urine. The newborn kidney is functionally non-efficient, has low glomerular filtration rate, a limited capacity to reabsorb sodium and a low ability to concen-trate urine (Velarde et al. 1995). Such non-efficient func-tional characteristics of the kidney extend after the postweaning period. For instance, adult urinary concentrationability is not reached until 6 weeks of age in the rat, anduntil 18 months after birth in humans (Rane et al. 1984,Aperia and Celsi 1992). In order to be functionally effi-cient, the kidney needs the full development of the entirenephron structure and also an adequate development ofthe collecting duct system. Growth and maturation of thekidney also continue after the completion of nephrogene-sis, and in rats it has been considered that nephrons reachterminal differentiation at the time of weaning (Nigam etal. 1996).

Much of the knowledge of how the metanephros de-velops to give rise to the permanent and functional kid-ney in higher vertebrates, comes from in vitro organ cul-ture systems and from fetal life. Although a lot of studieshave been performed about different aspects of the kid-ney postnatal development, the majority of them focusedon the development of the nephrogenic zone and/or aparticular cell subset. In the present work, we present adetailed analysis of the morphological changes that occur during the rat kidney postnatal development. Wealso establish the age at which the postnatal kidney losesits embryonic characteristics and acquires the adult orga-

M.G. Márquez · I. Cabrera · D.J. Serrano · N. Sterin-Speziale (✉)Laboratory of Cellular Biology and Histology, Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires,IQUIFIB-CONICET, Buenos Aires, Argentinae-mail: speziale@huemul.ffyb.uba.arTel.: +541-1-49648238, Fax: +541-1-49625457

N. Sterin-SpezialeDepartment of Biological Sciences, Cellular Biology and Histology,Faculty of Pharmacy and Biochemistry, University of Buenos Aires,956 Junin Street, 1st Floor, (1113) Ciudad Autónoma de Buenos Aires, Argentina

Anat Embryol (2002) 205:431–440DOI 10.1007/s00429-002-0262-9

O R I G I N A L A RT I C L E

María Gabriela Márquez · Isabel CabreraDiego Javier Serrano · Norma Sterin-Speziale

Cell proliferation and morphometric changes in the rat kidney during postnatal development

Accepted: 25 April 2002 / Published online: 29 June 2002© Springer-Verlag 2002

nization. We have studied the postnatal development ofthe cortex, the medulla and the papilla by combining immunohistochemistry and quantitative morphology. Westudied the distribution of PCNA-positive cells in orderto perform a topographic study of proliferating cells andthe binding of specific lectins to reveal changes in gly-cosilation in the developing kidney.

Materials and methods

Materials

Monoclonal antibody specific for Proliferating Cell Nuclear Anti-gen (PCNA) was purchased from Pharmingen (San Diego, Calif.,USA). Avidin-biotin based peroxydase detection kit (VectastainElite ABC Kit) was obtained from Vector Laboratories (Burlin-game, Calif., USA). 3,3’-diaminobenzidine (Sigma Fast DAB)were purchased from Sigma Chemical (St. Louis, Mo., USA). Biotin-conjugated Dolichos biflorus (DBA) and Griffonia simplicifolia I (Bandeiraea simplicifolia lectin I, BSL-I) were purchased from Vector Laboratories (Burlingame, Calif., USA).All other reagents and chemicals were purchased from variouscommercial suppliers.

Experimental animals and sampling

Preparation of kidney tissue to perform immunohistochemicalstudy

Weaning 2, 5, 7, 10, 11, 15 and 20-day-old rats of either sex and30 and 70-day-old male rats of the Wistar strain (closed colonyfrom the breeding unit kept at the animal facilities of the Instituteof Cardiology, Buenos Aires, Argentina) were used. Adult 70-day-old rats were used as controls. Experiments were performed using4 or 5 animals per group. Rats were sacrificed by decapitation, andboth kidneys were removed and placed in ethanol at 4 °C in orderto be processed by Sainte-Marie’s technique (Sainte-Marie 1962).Each kidney was cut in half through the pelvis along its longitudi-nal axis in order to expose cortex, medulla and papilla. Briefly, tis-sues were fixed in 95° ethanol pre-cooled at 4 °C, dehydrated infour changes of pre-cooled absolute ethanol, cleared by passingthrough three consecutive baths of xylene and embedded in paraf-fin at 56 °C. Tissue sections (4–5-µm thick) were placed on glassslides.

Immunohistochemistry

Immunohistochemical staining was performed on non-serial sec-tion. Paraffin was removed by gently swirling the slides in threeconsecutive baths of xylene, which was removed in two baths ofabsolute ethanol, two baths of 95° ethanol and three baths of phos-phate-buffered saline solution (PBS: 10 mM sodium phosphate,pH 7.5, 0.9% NaCl w/v in distilled water). Before incubation withthe monoclonal antibody PCNA, all tissue sections were incubatedwith 3% normal horse serum in PBS for 1 h at room temperatureto reduce non-specific binding. Incubation with the primary anti-bodies was performed overnight at 4 °C in horse serum containingbuffer, followed by three washes in PBS. Detection of positivecells was performed with an avidin-biotin based peroxydase detec-tion kit using 3,3’-diaminobenzidine as the chromogen. Endoge-nous peroxydase activity was quenched by incubating sections in0.3% H2O2/methanol for 30 min at room temperature.

For lectin staining, slides were incubated with the biotin-con-jugated DBA or BSL-I dissolved in PBS at a concentration of25 µg/ml for 45 min at room temperature, followed by a PBSwashing step. Detection of positive cells was performed with anavidin-biotin based peroxydase detection kit using 3,3’-diamino-

benzidine as the chromogen, avoiding the biotin-conjugated sec-ondary antibody step. Negative controls for the labeling proceduredescribed include deletion of the primary antibody or the lectinstep. The sections were counterstained with haematoxylin, mount-ed with balsam and examined by light microscopy. Two sectionsper animal were recorded. Two blinded investigators reviewed allslides. In order to perform the histological evaluation, sections ofthe kidney were stained with haematoxylin and eosin (H-E).

Morphometric analysis

Each kidney was cut in half through the pelvis along its longitudi-nal axis, and the measure of the minor axis of the kidney was taken from the capsule to papillary tip. We measured 4–8 renalsizes from each age studied (Fig. 7A).

The cortex width and the tubular epithelial cell height were de-termined by a morphometric analysis of randomized renal sectionsfrom all ages examined (4 or 5 rats in each group). Images wereacquired using a Nikon microscope, fitted with a SONY-SSC-DC50A video camera controlled by a Pentium personal computer.The morphometric measurements were performed with the SigmaScan software package (Jandel Scientific Software).

To estimate the cortical wide, a 4X objective was used and thelength of the cortex was measured by extending a line from theouter capsule to the parietal layer of the most juxtamedullary renalcorpuscles (Fig. 7A). A minimum of 8 values (cortex length inmillimeters) were randomly measured in each slide. In order to es-timate the size of individual cells, a 100X objective was used andthe height of cells from papillary collecting ducts, cortical proxi-mal and distal convoluted tubules were measured by extending aline from the basement membrane to the apical border of each cell(Fig. 7B). A minimum of 10 values (cell height in µm) was ran-domly measured in each tubule.

Statistical analysis

Statistical analysis was done using the GraphPad Prism software.Significance was considered acceptable at the 5% level (P<0.05).

Cell proliferation. Results are expressed as the mean number ofPCNA+ cells (nuclear labeling) ± standard error of the mean(SEM), found in ten fields at magnification X1,000 per tissue section, 4 or 5 animals per group and two sections per animal.ANOVA and Dunnett test.

Morphometric analysis. (a) Kidney minor axis and cortex width.Results are expressed as X ± SEM, 4–15 animals per group. ANOVA and Tukey Kramer test. (b) Cell height. Results are ex-pressed as X ± SEM, 3–5 animals per group. ANOVA and Dunnettmultiple comparison test.

Results

Postnatal development of kidney cortex

Histological sections of rat kidneys of various ages wereanalyzed to study the postnatal development of the renalcortex. Adult rats were used as control.

At 70 days of age, the renal cortex from the subcapsu-lar to the juxtamedullar region is densely packed withglomerulus, proximal and distal convolutes tubules(PCT, DCT) of mature nephrons and collecting duct(CD) tubules. As it is well known, the total cellular levelof PCNA in adult rats is relatively constant through thecell cycle and it is tightly associated with the nucleus only in S-phase (Celis and Celis 1985, Bravo and

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MacDonald-Bran 1987). In the present study, nuclearPCNA immunostaining is not found in adult renal cor-tex, indicating no detectable cells in proliferating state,while the presence of cytoplasmic PCNA is evident intubular epithelial cells (not shown).

In the neonatal period, from day 2 to day 7, threewell-defined cortical zones are observed (Fig. 1A). Thefirst zone, which is nephrogenic, and where all the im-mature forms of renal developmental stages as conden-sates/vesicles, comma-shaped and S-bodies are present,

is subcapsulary located. In this zone, blind ends of devel-oping CD are also present. The second zone, whereformed glomeruli surrounded by PCT and DCT are lo-cated, is juxtamedullar. The third zone, which extendsfrom the capsula up to the medulla, and which is locatedin the zone where in the adult are the medullary rays, isvery densely occupied by uninduced, disorganized cells.

At postnatal day 10, most nephrons are immature.Pre-glomerulus are evident and blind ends of branches ofureteric buds (UB) are still present (Fig. 1B, C). How-ever, a higher degree of nephrological maturation is ob-served in the juxtamedullary zone, where nephrons aremore differentiated than the subcapsular ones. At postna-tal day 15, there is no further branching of UB. At wean-ing, the cortical zone has the same morphological aspectas that observed in adult rats (not shown).

To detect the cell type that is in the proliferating state,we examined PCNA immunostained sections. From 2 to15 days of age most of the PCNA staining is associatedto the nucleus, reflecting high proliferating state of thecells (Fig. 1D, E). Fig. 2 reports the quantification of nu-clear positive PCNA cells. High density of PCNA posi-tive cells is observed in the subcapsular nephrogeniczone at day 2 declining with age (Fig. 2A). A very high

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Fig. 1 A Cortex of a 5-day-old rat showing the subcapsular nephro-genic zone (H-E). B Low magnification of renal cortex from a10-day-old rat indicating an area shown at higher magnificationin C: DBA+ blind ends of a branch of the ureteric bud (asterisks).Lectin-binding, peroxidase method. D Low magnification of renalcortex from a 7-day-old rat immunostained for PCNA indicatingan area shown at higher magnification in E. E High amount ofproliferating cells (arrow) in the zone known in the adult kidney as medullary rays. F, G Cortex of adult rat showing DBA (F)and BSL-I (G) binding. H, I Cortex of a 2-day-old rat showingDBA (H) and BSL-I (I) binding Lectin-binding, peroxidase method(UB ureteric bud, S S-bodies, V vesicles, MR medullary rays, G glomerulus, P proximal convoluted tubules, D distal convolutedtubules, CD collecting ducts). A, B and D ×100, C, E-I ×400

amount of proliferating cells is also observed at day 2 inthe zone classically known in the adult kidney as medul-lary rays. In this zone, proliferating cells are maintainedhigh up to 15 days of age, dramatically decreasing there-after (Fig. 2B). At day 5, a decrease in proliferating cellsis observed in glomerulus as well as in tubular cells, in-creasing at day 7 (Fig. 2C, D). From day 10 onwards, adefinitive decline is observed in glomerular cells, whiletubular cells still proliferate, declining thereafter, andconserving a low degree of proliferation in the adult.Proliferation in interstitial cells decreases with the ageand shows three steps, one after day 5, and other one after day 15 and the last one after day 30 (Fig. 2E).

To examine the developmental changes in glycosyla-tion, we tested the binding of lectins DBA and BSL-I. Lec-tin DBA binds specifically to N-acetyl galactosamine–α1,3 N-acetyl galactosamine and lectin BSL-I to α-N-acetylgalactosamine and α-D-galactose (Damjanov 1987). At allages examined, portions of the nephron differ from one an-other with regard to their lectin binding properties. In theadult rats (Fig. 1F, G), the glomerulus are negative forDBA and there are few weak positive cells for BSL-I bind-ing. Both PCT and DCT cells are positive for DBA. How-ever, the intensity of binding is different, being stronger for

DCT and weaker for PCT cells. Both convoluted tubularcells are positive for BSL-I, showing no differences in theintensity of binding. Cortical CD cells are very stronglypositive for DBA binding and completely negative forBSL-I. No DBA or BSL-I reactivity has been observed inthe interstitial compartment.

In developing kidney, lectin binding led to differenti-ating the external highly immature zone from the juxta-medullar zone (Fig. 1H, I). The immature nephrogenicstructures of the subcapsular zone and the subcapsularepithelium are always negative for DBA and BSL-Ibinding, while the internal, already induced tubularstructures slightly bind lectins. The glomerulis are nega-tive for DBA binding from day 2 up to adulthood andthere are few weak positive cells for BSL-I binding fromday 5 and thereafter. Very early in the postnatal period,PCT are indistinguishable from DCT either by morphol-ogy or lectin staining. Both tubules are weakly DBA andBSL-I positive but at postnatal day 20, the intensity ofbinding begins to acquire the adult pattern. Cortical CDcells are very strongly positive for DBA binding at allages examined even in the stage of blind end of UB(Fig. 1H). At postnatal day 2, UB cells present apicalBSL-I reactivity, which disappears thereafter (Fig. 1I).

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Fig. 2 Quantitative assessmentof proliferating cells in renalcortex during the postnatal development (# P<0.05 thanvalue at 20 days, * P<0.05than value at 70 days)

No lectin reactivity has been observed at any age in theinterstitial compartment.

Postnatal development of kidney medulla

Two different regions are observed in the renal medullafrom adult rats: the outer medulla (next to the cortex) andthe inner medulla (next to the papilla). The adult outermedulla can be divided into outer strip and inner strip

(Fig. 3A). Very few tubular epithelial and interstitial pro-liferating cells are observed in adult renal medulla.

At postnatal day 2 (Fig. 3B), the medullary region re-veals extreme disorganization and it is not possible todistinguish outer from inner medulla. In a panoramicview, an abundant interstitium with some islets of tubu-lar structures is observed (Fig. 3C). Interstitial cellsshow different sizes and shapes, denoting a high degreeof undifferentiation. Heterogeneity is also observed intubular structures, since cells with extremely big nucleiand others with intermediate and small nuclei coexist inthe structures. The number of cells in each tubule is alsodifferent. Most of the tubules consist of 4 or 6 cells, butthere exists structures with up to 8 cells.

Very early in the postnatal developmental period inthe renal medulla, there are DBA positive UB/CD tu-bules surrounded by DBA negative smaller tubular struc-tures that bind BSL-I lectin (Fig. 3D–F). Peritubularspaces surrounding the UB/CD are observed at postnatalday 5 and 11. At day 7, the tubules are bigger and the in-terstitium looks more organized. At postnatal day 10, theinner zone and an outer zone start to be differentiated.Still at day 15, renal medulla is not completely devel-oped and no distinction between outer and inner strip of

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Fig. 3 A Medulla of adult rat showing the outer (OS) and the inner(IS) strip of the outer zone. B Low magnification of renal medullafrom a 2-day-old rat indicating an area shown at higher magnifica-tion in C. C An abundant interstitium with some islets of tubularstructures (arrows) is observed (H-E). D Low magnification of re-nal medulla from a 5-day-old rat (H-E) indicating areas shown in Eand F. E DBA positive UB/CD surrounded by peritubular spaces(asterisks). F tubular structures surrounded by UB/CD BSL-I nega-tive (lectin-binding, peroxidase method). G Area of the medullaof a 2-day-old rat (box in C) showing nuclear PCNA expressionin the tubular (arrowhead) and in the interstitial compartment (arrow). Peroxidase method. H, I Medulla of adult rat showingDBA (H) and BSL-I (I) binding. Lectin-binding, peroxidase meth-od (UB ureteric bud, CD collecting duct, tLH thin limb of Henle,TLH thick limb of Henle, T tubular structures). A, B and D ×100,C, E-I ×400

the medullary outer zone is observed. The greatestchange occurs between postnatal day 20 and 30. At day30, there is a clear distinction between the outer and theinner strip of the outer zone. The renal medulla acquiresthe morphological appearance of the adult medulla aslate as postnatal day 30 (not shown).

A high number of nuclear PCNA positive cells is foundin the tubular and interstitial compartment at 2 days of age(Figs. 3G, 4A, B). At day 5, both tubular and interstitialcells in S-phase fall dramatically (Fig. 4A, B). At day 15,the highest number of proliferating cells is observed bothin the outer and in the inner medulla, which reflects elon-gation of the straight portion of the PCT and DCT, givingrise to the outer strip of the outer zone of the medulla(Fig. 4A, B). Peritubular space surrounding UB/CD is ob-served at day 11, concomitant with a decrease in the num-ber of proliferating cells (Fig. 4A, B). The inner medulla,which is not apparent until day 11 of postnatal life, showsthe highest number of proliferating cells at day 15, de-creasing thereafter up to adult life (Fig. 4A, B).

The adult glycosylation pattern is shown in Fig. 3H, I.The medullary CD cells are positive for DBA and nega-

tive for BSL-I binding. Both thin (tLH) and thick (TLH)limb of Henle’s cells are positive for BSL-I while weakDBA binding is only observed in TLH cells. The intersti-tial compartment is BSL-I positive and DBA negative.CD cells are positive for DBA and negative for BSL-Ibinding at all ages examined and the opposite is ob-served in tLH. A weak BSL-I reaction in the apical zoneof TLH cells is observed. BSL-I reactivity appears in theinterstitial compartment in the inner medulla by postna-tal day 30; but no positive cells are observed in the outermedulla at this age. As the medulla matures, more inter-stitial cells in both medullary zones are positive for thislectin. In contrast, all interstitial cells are negative forDBA binding at all ages examined (not shown).

Postnatal development of kidney papilla

Histologically, the renal papilla from adult rats is formedby CD, tLH, interstitial cells and the papillary surfaceepithelium. It is possible to observe two different re-gions: the proximal papilla (next to the medulla) and the

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Fig. 4 Quantitative assessmentof proliferating cells in renalmedulla during the postnataldevelopment. (* P<0.05than value at 70 days)

Fig. 5 Quantitative assessmentof proliferating cells in renalpapilla during the postnatal development. Positive cellsin the proximal papilla were recorded when its value differsfrom those from distal papilla.(* P<0.01 than value at70 days)

distal papilla. In adult renal papilla, no proliferating cellsare observed (Fig. 5A–C).

Three compartments are distinguishable in the devel-oping papilla, namely the tubular compartment constitut-ed by UB/CD and tLH, the interstitial compartment andthe papillary surface epithelium. Since postnatal day 2, itis possible to observe a region next to the medulla where

mature CD and tLH are located and a zone of the tipwhere new UB/CD form by invagination of the surfaceepithelium (Fig. 6A). By contrast, tLH with the same ap-pearance are present in both zones from day 2 to adult-hood. From postnatal days 2 to 10, the papilla is denselypacked with CD, tLH and capillaries but the papillary in-terstitium is small. At postnatal day 15, the interstitiumbecomes much more abundant, containing a heterogene-ous population of interstitial cells.

At postnatal day 2, a high number of proliferatingcells is observed in the tubular compartment, in the inter-stitium and in the epithelium lining the papillary surface,decreasing thereafter and being absent in adult rats(Figs. 5A–C, 6B). At day 10, a striking increase in thenumber of cells in S-phase is observed in the interstitialcompartment (Fig. 5B). This observation can explain thegrowth of the interstitial compartment that is observed at15 days of age. The number of proliferating cells foundin the tubular structures in the proximal papilla from day7 to 30 is higher than that found in the distal papilla(Fig. 5A). This suggests that the elongation of papillarytubules occurs in the proximal papilla where the transi-tion between medulla and this last zone is located.

At postnatal days 5, 11 and 20, a peritubular spacesurrounding UB/CD is observed (Fig. 6C, D). After day

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Fig. 6 A Low magnification of renal papilla from a 2-day-old ratindicating an area shown at higher magnification in B. Notethe invagination of the surface epithelium (asterisks). B NuclearPCNA immunolabeling of tubular (arrowhead) and interstitialcells (arrow). Peroxidase method. C Low magnification of renalpapilla from a 20-day-old rat indicating an area in the distal papil-la at higher magnification in D. D DBA positive UB/CD surround-ed by peritubular spaces (asterisks). Lectin-binding, peroxidasemethod. E Low magnification of renal papilla from an adult rat indicating an area at higher magnification in F. F strong DBA-positive CD and DBA negative tLH and interstituim. G Papillafrom an adult rat showing BSL-I staining of tLH and interstitium.No reactivity is detected in CD. H Low magnification of renal papilla from a 15-day-old rat indicating an area shown at highermagnification in I. I Distal papilla from a 15-day-old rat showingfew BSL-I positive CD cells (arrows). Lectin-binding, peroxidasemethod. (UB ureteric bud, CD collecting duct, I interstitium, tLH thin limb of Henle, SE surface epithelium). A and D ×100, B,F-H ×400, C and E ×40, I ×1000

20, the histological appearance of the papilla is similar tothat found in the adult.

At all ages examined, CD and surface papillary epi-thelium cells are strongly positive for DBA binding andwe never observed DBA reactivity in tLH and interstitialcells (Fig. 6E, F). In adult rats, CD from distal and proxi-mal papilla and the surface epithelium are BSL-I-nega-tive (Fig. 6G). However, at postnatal day 2, when newUB/CD are undergoing a process of formation, both thesurface epithelium and UB/CD cells are apically labeledwith BSL-I. Such BSL-I positive epithelium is observedonly in the invaginating cells of the distal papilla untilpostnatal day 20 (Fig. 6H, I). It is important to note thatthe appearance of the epithelium covering the papillarysurface is similar to that observed in the epithelium ofthe CD, at all ages examined. Moreover, both epitheliabind the DBA lectin, whose specificity to the UB/CD iswidely accepted (Laitinen et al. 1987). This observationmay constitute further experimental evidence to supportthe knowledge that the UB and thus the kidney CD

system are derived from the surface papillary epithelium.tLH cells bind lectin BSL-I but not DBA at all ages examined and from day 11 of postnatal life, all papillaryinterstitial cells are BSL-I positive.

Morphometric analysis

As shown in Fig. 7C the renal size, estimated by measur-ing the kidney minor axis (Fig. 7A), increases graduallyfrom day 2 to day 70. No significant difference is ob-served in the age-related increase in cortex size from 2 to20 days of age, but there is a significant increase fromday 30 to day 70 (Fig. 7C).

Early in the postnatal development, it is very difficultto distinguish the boundary between the medulla and thepapilla by histological analysis. Thus, we have analyzedthe medulla-papilla region as a whole. We observed thatthe medulla, as well as the papilla, increases in size sincebirth. In order to estimate the individual cell sizes, we

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Fig. 7 Scheme of the measur-ing procedure of the renal minor axis, cortical width (A)and cellular height (B). C Kidney width: ** P<0.001from all the remaining groups,* P<0.001 from all the remain-ing groups except betweenthem. Values at 5, 7 and10 days P<0.001 from the re-maining groups except betweenthem and (#) P<0.001 from allthe remaining groups exceptfrom postnatal day 5. C Cortexwidth: * P<0.001 from allthe remaining groups andbetween them, except 20 vs 30 and 30 vs 70, P<0.01. D PCT cell height: * 2, 7and 20 vs 70, P<0.05 and 5, 10and 15 vs 70, P<0.01). E DCTcell height: * 2, 5, 7, 10-11and 15 vs 70, P<0.05 and 20 vs70, P<0.01. F CD cell height: * 5 vs 70, P<0.05

have determined the individual tubular epithelial cellheight of papillary CD, PCT and DCT (Fig. 7B). Wehave observed that the most rapid increase in cell sizeoccurs in the postweaning period for PCT and DCT cells(Fig. 7D, E). By contrast, the size of CD cells in the pre-weaning period is quite similar to that observed in adultrats (Fig. 7F). However, a remarkable decrease in cellsize is observed at postnatal day 5. After day 5, the cellu-lar size begins to increase again (Fig. 7F).

Discussion

Kidney embryonic development has been described as atwo-stage process. The first stage involves inductive in-teractions between UB and metanephric mesenchyme, thesecond stage is marked by the rapid division of tubularepithelial cells, which takes place in the subcapsularnephrogenic zone (Grossens and Unsword 1972; Saxen1987). As shown in the present report, the first inductivestage described by Grossens occurs until around postnatalday 10, when it is possible to observe blind ends ofUB/CD branches underneath the capsule. The secondstage, when an active process of cortical cell proliferationand differentiation takes place, is present as late as day 20of postnatal life, being the earliest age when kidney cor-tex acquires the conformation of the mature adult cortex.

The high degree of proliferation of cells from thenon-organized subcapsular and medullary ray zones cansuggest that the cells for the postnatal formation of newnascent nephrons stem from the subcapsular zone andalso from the medullary ray zone, while proliferation inalready formed tubules, glomeruli and interstitium prob-ably accounts for the further tubular elongation andgrowing process.

The renal medulla is the most immature kidney zoneat birth; it matures later than the cortex and the papillaand displays the greatest morphological changes duringthe postnatal development. In newborn rats (day 2) itdoes not look like a kidney zone but like islets of tubularstructures surrounded by interstitium. Cellular divisionoccurs in already-organized tubules and probably servesfor tubular expansion. Concomitant with tubular prolifer-ation, the medulla expands as CD and loops of Henlelengthen, although a distinct inner and outer medullacannot be distinguished before postnatal day 10. Afterday 10, tubular proliferation continues, accounting forouter medullar expansion and the earliest age when theouter and inner strip of the medullary outer zone can beclearly distinguished is at day 30 of postnatal life. Thisobservation is consistent with the proliferative process,which is higher in tubular epithelial cells from the outermedulla than in those from the inner medulla.

The degree of proliferation is low in the papillary tubules. This observation together with the active epithe-lium invagination process led us to suggest that the post-natal newly formed CD system stems from the papillaryexternal epithelium. By contrast, tubular cell prolifera-tion must account for the elongation process, which in

turn is more active in the proximal than in the distal papilla, denoting that papillary growth occurs mainly byproximal papillary region expansion.

A relationship between ECM remodelation and tubu-lar elongation has been described for UB branching andgrowth during kidney development (Pohl et al. 2000). Interestingly, two and three events of ECM remodelingtake place around medullary and papillary CD, stoppingat day 11 and at day 20 respectively, during postnatal development. The degradation of the ECM surroundingCD would probably be required to provide space for ductexpansion since the number of cells found in a cross sec-tion through the CD in the medulla and papilla in the dayof remodeling is lower than that observed thereafter.

As it is known, an organ can increase in size at thecellular level due to an increase in cell number (hyper-plasia; Jakobson et al. 1987) by increased proliferationor decreased apoptosis, or by an increase in individualcell size (hypertrophy). However, we are not consideringthe contribution of the normal cell death to the postnatalkidney development, which has been very well describedby others (Coles et al. 1993). Aperia (Aperia 1992) hasreported that in the rat kidney, the most rapid cell multi-plication occurs in the preweaning period, whereas themost rapid increase in size occurs in the weaning period.Although in our morphometric analysis we demonstratedthat there is a gradual increase in the total kidney sizeduring development, its size is temporally different foreach kidney zone and due to different structures. The in-crease of the cortex occurs after day 20 and it is mainlydue at least to tubular hypertrophy, while medullary andpapillary increase in size is continuous from day 2 to day30 while the increase in size of the medulla is due to tu-bular hyperplasia, it first occurs by tubular elongation inthe papilla, though interstitial expansion predominatesthereafter.

Changing patterns of lectin binding reflect sequentialmasking of embryonic glycoconjugates, growth-relatedmodification of cell components, and functional matur-ing cells (Damjanov 1987). To examine the establish-ment of an adult type of glycosylation, we have com-pared the binding of lectins DBA and BSL-I during therenal postnatal development with the one in the adult.Our present results demonstrate that, although early indevelopment, cells assume different phenotypes and be-come committed to a specific cell lineage, protein glyco-sylation remains undifferentiated until the postnatal peri-od and the establishment of an adult type of glycosyla-tion is different in the various zones of the kidney andfor the different cell types. At birth (day 2), lectin bind-ing is only observed in the juxtamedullar zone where or-ganized tubular structures are located, while the subcap-sular nephrogenic structures in the cortex are negativefor the binding of lectin DBA and BSL-I. As kidneymaturation progresses, PCT and DCT acquire glycocon-jugates that progressively bind both lectins.

Previous reports have shown that BSL-I lectin doesnot react with CD cells in adult rats (Grupp et al. 1997)but BSL-I reactivity has been reported in CD cells from

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mouse embryonic kidney (Laitinen et al. 1987). In thepresent report, CD from adult rats is also BSL-I nega-tive. However, the cells of new UB/CD represented byinvagination of the surface epithelium are BSL-I-posi-tive, while no labeling is observed in more mature CDcells. As a result, it is likely that the surface epithelium,which suffers from such invagination to form UB/CD,will keep embryonic glycoconjugates that bind BSL-Ithat are lost or masked by other glycoconjugates as mat-uration progresses. Taking into account the results pre-sented above, we are tempted to suggest that the sequen-tial acquisition or loss of a special kind of glycoconju-gates could be considered as a marker of a differentiatedstate during the postnatal renal development.

Interestingly, tLH, which are mainly located in the papilla at birth, are morphologically indistinguishablefrom the one in the adult and they are also BSL-I-posi-tive, denoting mature glycosylation pattern. It seems tous that tLH probably constitutes the most mature neph-ron structure at birth. Consequently, we are tempted tospeculate that such limbs of Henle could be the result ofthe earliest inducing process accounted by the embryonicUB branching.

The comparative study presented in this work clearlydemonstrates that the postnatal development of the ratkidney is a highly synchronized process where the pat-tern of growth and cell differentiation is characteristicfor each cell type and for each zone of the kidney, givingrise to the high anatomical and functional compartmen-talization of this organ. The cortex and the papilla ac-quire the morphological appearance of the adult kidneybefore the medulla does. Consequently, the medulla re-mains at the highest degree of immaturity, among thekidney zones, for a relatively long postnatal period. Thisfact can explain the liability of this particular kidneyzone to pathological conditions as is the specific necrosisinduced by choline deficient administration of postwean-ing rats (Monserrat 1974, Montes de Oca 1980).

Acknowledgements The authors thank Mr. Valentin Serra andVerónica Castro for technical assistance. This investigation wassupported by grants from CONICET, University of Buenos Airesand the Minister of Health of Argentina.

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