1 Institut für Anatomie, Charité, Humboldt Universität, D-10098 Berlin, Germany; 2 University of Colorado Health Sciences Center and Veterans Affairs Medical Center, Denver, Colorado; 3 Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1002 Lausanne, Switzerland; 4 University of Arkansas, Little Rock, Arkansas; and 5 Institut National de la Santé et de la Recherche Médicale, Unité 478, Unité d'Enseignement et de Recherche Xavier Bichat, F-75877 Paris Cedex 18, France
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ABSTRACT |
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During the past several years, sites of expression of ion
transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were
defined by mapping the expression of sodium transport pathways during
nephrogenesis in the rat. Combined in situ hybridization and
immunohistochemistry were used to localize the
Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the
thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger
(NaCa), the epithelial sodium channel (rENaC), and 11-hydroxysteroid
dehydrogenase (11HSD). The onset of expression of these proteins began
in post-S-shape stages. NKCC2 was initially expressed at the macula
densa region and later extended into the nascent ascending limb of the
loop of Henle (TAL), whereas differentiation of the proximal tubular
part of the loop of Henle showed a comparatively retarded onset when
probed for NaPi2. The NCC was initially found at the distal end of the
nascent distal convoluted tubule (DCT) and later extended toward the
junction with the TAL. After a period of changing proportions,
subsegmentation of the DCT into a proximal part expressing NCC alone
and a distal part expressing NCC together with NaCa was evident. Strong
coexpression of rENaC and 11HSD was observed in early nascent
connecting tubule (CNT) and collecting ducts and later also
in the distal portion of the DCT. Ontogeny of the expression
of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data
present a detailed analysis of the relations between the anatomic
differentiation of the developing renal tubule and the expression of
tubular transport proteins.
nephrogenesis; ion transport; tubular segmentation; mineralocorticoids; rat kidney
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INTRODUCTION |
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THE KIDNEY plays a central role in fluid and electrolyte homeostasis. Several genes that participate in sodium reabsorption along the nephron have recently been cloned; human mutations of these genes cause alterations in blood pressure (27, 37, 39). These genes encode ion transport proteins such as exchangers (36), cotransporters (16), and ionic channels (7). From an anatomic standpoint, the nephron may be divided into proximal and distal portions. The proximal tubule comprises convoluted and straight portions. The distal tubule comprises the medullary and cortical thick ascending limbs (TAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical and medullary collecting ducts (CCD and MCD, respectively). Each of these segments has unique functional properties that have been investigated using physiological techniques (9, 11, 14, 41).
Recent molecular studies on the distribution of sodium transporters
have more precisely defined nephron segmentation. The Na/H exchanger
(NHE3; Ref. 4) and the Na-Pi
cotransporter type 2 (NaPi2; Ref. 10) are both expressed by cells of
the proximal tubule. NHE3 contributes importantly to Na and bicarbonate
reabsorption, whereas NaPi2 contributes to Na and phosphate
reabsorption. Although proximal tubular sodium absorption may adapt to
states of volume expansion or contraction, changes in urinary sodium
and chloride excretion reflecting variations in salt intake are mainly
governed by distal tubular functions. In the ascending limb of the loop of Henle, expression of the electroneutral, furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) has been found in the TAL, the macula
densa, and a portion beyond the macula densa (32). A structurally
related thiazide-sensitive Na-Cl cotransporter (NCC) was localized to
the DCT (2, 33, 35). The amiloride-sensitive epithelial sodium channel
(rENaC; Ref. 7) has been shown to be expressed by distal nephron cells,
together with 11-hydroxysteroid dehydrogenase type 2 (11HSD) and
mineralocorticoid receptors (5, 6, 25, 26). Recent evidence points out
that mutations in these genes lead to disorders of sodium and
volume homeostasis (37). Mendelian forms of human hyper- or hypotension
have been linked, using molecular genetics, to mutations of a number of these genes, including NKCC2, NCC, rENaC, and 11HSD (27,
39).
During development, the mammalian nephron and collecting duct (CD) system undergo complex, transient patterns of structural differentiation on their way to segmental specialization (12, 30, 31). Despite extensive study, however, it has been difficult to identify developmental regions of transition, especially the junction between the ureteric bud and the nephrogenic blastema. This study examines the expression patterns of sodium transport proteins in the developing rat nephron using high-resolution histochemical techniques. By recording the changing patterns of expression of these proteins in the maturing epithelia, new aspects of the genesis of segmentation of the renal tubule are suggested. The definition of a normal ontogenetic expression of ion transport pathways is thought to provide a basis for the study of transport disorders in later life which may originate during ontogeny.
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MATERIALS AND METHODS |
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Animals. For perfusion fixation, Sprague-Dawley rats at postnatal stages of 1, 3, 4, and 8 days were anesthetized with ether. For immunocytochemistry and in situ hybridization, kidneys were perfused via cannulation of the left cardiac ventricle. Perfusion was performed using freshly prepared 3% paraformaldehyde (PFA) in PBS at pH 7.4 for 5 min. To protect the tissues from freezing artifacts, kidneys were subsequently rinsed in situ with a sucrose-PBS solution adjusted to 800 mosmol/kg. Kidneys were then removed, cut into slices, and shock-frozen in liquid nitrogen-cooled isopentane. For fine structural morphology, animals were anesthetized with an intraperitoneal injection of Nembutal (40 mg/kg body wt), and kidneys were retrogradely perfusion fixed via cannulation of the abdominal aorta using a solution of 3% glutaraldehyde and 1.5% PFA buffered with PBS, pH 7.4. Kidneys were then removed and processed for Epon embedding.
Morphology. Semithin sections (1 µm) were cut and stained in Richardson's solution. Slides were viewed in a Leica DMRB microscope. For electron microscopic studies, ultrathin sections were analyzed after contrasting in uranyl acetate and lead citrate. Sections were examined with a Zeiss EM 900 electron microscope.
In situ hybridization. For generation of a riboprobe to the NCC, a 712-bp partial cDNA fragment of mouse NCC was used (33). A riboprobe to NKCC2 was prepared from a 375-bp partial cDNA fragment of the mouse NKCC2 (32). The NaPi2 probe was prepared from a cDNA fragment that was generated by RT-PCR comprising the sequence from position 1561 to 2355 of rat NaPi2; the identity of the cDNA probe was verified by sequencing. An NAD-dependent 11HSD probe was generated from a 248-nucleotide cDNA fragment of rat kidney 11HSD (5). These fragments were all subcloned into the EcoR V site of pBluescript KS+ vector (Stratagene, La Jolla, CA). To generate sense and antisense riboprobes for 11HSD, the vector was linearized either by Hind III or by BamH I, respectively. For generation of NCC, NaPi2, and NKCC2 riboprobes, PCR fragments comprising the respective insert and specific transcription promoters were synthesized using vector-specific complementary oligonucleotides. RNA probes were synthesized and labeled by in vitro transcription using digoxigenin (DIG)-labeled UTP and T3 or T7 RNA polymerases (Boehringer, Mannheim, Germany) to obtain either sense (control) or antisense transcripts, respectively.
In situ hybridization on 7-µm cryostat sections was essentially done as described (32). Briefly, DIG-labeled probes were used at concentrations of 2 to 8 ng/µl of hybridization mixture. Hybridization was performed at 40°C for 18 h. Slides were washed at 40°C for 30 min in 1× SSC containing 50% formamide, followed by two washes for 30 min in 0.4× SSC containing 50% formamide and two subsequent washes for 30 min in 0.1× SSC containing 50% formamide. Subsequently, slides were rinsed twice in 0.5× SSC at room temperature for 10 min, followed by a rinse in 0.2× SSC for 10 min and another two rinses in buffer I (100 mM Tris · HCl and 150 mM NaCl at pH 7.5). After quenching of nonspecific antibody binding sites with blocking medium (2% normal sheep serum, 0.5% BSA, 3% Triton X-100 in buffer I), sheep anti-DIG-alkaline phosphatase conjugate (diluted 1:500 in blocking medium) was administered to the sections. After removal of excess antibody, the substrate (nitroblue tetrazolium with 5-bromo-4-chloro-3-indolyl phosphate) for alkaline phophatase was added.
In situ hybridization for detection of rENaC mRNA was performed with
radiolabeled riboprobes. To prepare riboprobes part of the
3'-untranslated region to the - and
-subunits of rENaC
(corresponding to nucleotides 2150 to 2463 for the
-subunit, and
2470 to 2911 for the
-subunit) were subcloned into pBluescript
KS+ vector and linearized with
BamH I or
Kpn I. Probes were synthesized using a
T3/T7 in vitro RNA synthesis kit (Promega) in the presence of
35S-labeled UTP (Amersham).
Hybridization was essentially done as described (13). In brief,
cryostat sections were postfixed in 4% PFA, treated with proteinase K,
acetylated, covered with the hybridization mixture, and incubated
overnight at 50°C. For simultaneous detection of NKCC2 and NaPi2
mRNA, the two cRNA probes were combined in the same hybridization mix
and applied to the same section. Washing was done with 5× SSC and
10 mM dithiotreithol (DTT) at 50°C for 30 min, followed by a
high-stringency wash in 50% formamide, 2× SSC, and 0.1 M DTT at
65°C for 20 min, then several washes in NaCl-Tris-EDTA (0.5 M NaCl,
5 mM EDTA, and 10 mM Tris · HCl, pH 7.4) at 37°C.
After subsequent RNase treatment, sections were rinsed in 0.1×
SSC for 15 min, dehydrated, and dried. For autoradiographic detection,
slides were dipped in Kodak NTB2 photoemulsion (Kodak, Rochester, NY),
dried, and exposed at
20°C for 2 wk. Prior to viewing,
slides were counterstained with hematoxylin-eosin.
Generally, control experiments were done using sense probes for the respective cRNA probes; throughout all experiments, the controls yielded uniformly negative results.
Immunocytochemistry. For
immunolabeling, polyclonal antibodies, which had been characterized
previously (5, 13, 25, 36), were used. Antibody to NCC was generated
against a fusion protein containing the entire
NH2-terminal tail of mouse NCC
(5). Antibody to 11HSD was generated against a fusion protein
corresponding to human 11HSD (25). For generation of antibodies to the
-,
-, and
-subunit of rENaC, fusion proteins from the
NH2-terminal end of
-rENaC
(amino acids E10 to F77), from the COOH-terminal end of
-rENaC
(amino acids G559 to E636), and from the COOH terminus of
-rENaC
(amino acids A570 to L650) were used (13). The strongest immunoreactive
signal in tissue sections was obtained with anti-
antibody. All
these antibodies were generated in rabbits. Antibody against the Na/Ca
exchanger (NaCa) was generated in guinea pig against a fusion protein
of rabbit NaCa (36). Antibodies to NaCa and rENaC were used at a
dilution of 1:100 in PBS, antibody to NCC was used at a dilution of
1:200, and antibody to 11HSD was used at a dilution of
1:200-1:1,000. Antibody to band 3 was used as a control marker for
type A intercalated cells (type A IC, dilution 1:3,000; antibody was
kindly provided by Dr. Jöns, Berlin, Germany).
Immunohistochemical staining was performed on cryostat sections of 4-7 µm thickness. Sections were pretreated with 2% BSA in PBS, pH 7.4, for 30 min and incubated with the primary antibody for 2 h at room temperature and then at 4°C overnight. After thorough rinsing in PBS, bound antibody was detected by a 1-h incubation with Cy2-conjugated goat anti-guinea pig IgG serum, diluted 1:60 in PBS and Cy3-conjugated goat anti-rabbit IgG serum (DIANOVA, Hamburg, Germany), diluted 1:250 in PBS, or by the peroxidase antiperoxidase method (PAP), respectively. Detection by PAP was done by a 30-min incubation of pig anti-rabbit IgG serum (DAKO, Glostrup, Denmark) diluted 1:20 in PBS followed by a 30-min incubation with the PAP complex (DAKO). Signal was generated with 0.1% diaminobenzidine and 0.02% H2O2 in PBS.
For combined application of in situ hybridization and immunohistochemistry, specific antibody was either administered in parallel with the anti-DIG-alkaline phosphatase conjugate and marked with Cy2- or Cy3-labeled secondary antibody prior to detection of the riboprobe, or immunohistochemistry was conducted as described for single antibody labeling when detection of riboprobe was already finished. For triple labeling using a cRNA probe and two different antibodies, in situ hybridization was performed and, after signal generation, a mixture of antibodies from different animals was applied. After washing with PBS, suitable secondary antibodies coupled to different fluorochromes were applied.
Controls for immunoreactivity were done by replacing specific antibody with preimmune sera (when probing for rENaC and NCC) or with PBS (when probing for NaCa and 11HSD).
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RESULTS |
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Identification of nephrogenic stages. In rat, the centrifugal pattern of fetal nephrogenesis is continued after birth for approximately 6 days so that all the distinct nephrogenic stages may be studied in the kidneys of newborn rats. The development of the nephron has been divided into five clearly distinguishable stages (24). Stage I corresponds to the renal vesicle, stage II to the S-shaped body. Stages I and II are located within the nephrogenic zone, which is the region of nephron induction directly beneath the renal capsule. In stage III, the glomerulus is oval or spherical and the typical glomerular capillary loops are formed. The glomerular visceral epithelial cells are still closely apposed to each other with narrow intercellular spaces. At this stage, the first signs of epithelial segmentation of the tubule become obvious at the ultrastructural level. The loop of Henle does not yet descend past the medullary pole of the glomerulus. In stage IV, the glomerulus has become larger and capillary loops are more numerous than in the preceding stage. The primitive loop of Henle has by then descended beyond the medullary pole of the glomerulus but still lacks a tubular lumen. Later in stage IV, the "primitive loop" transforms into the "immature loop" of Henle with a patent lumen (30). At this level, the proximal tubular epithelium carries the typical brush border, and all the segments defined in the adult nephron can be recognized at the gross structural level in stage IV. In stage V, the nephron is approaching mature morphology.
Expression of NKCC2. In the mature
nephron, NKCC2 is expressed by cells of the TAL including the macula
densa and a short post-macula segment (32). During early postnatal
stages (days 1,
3, and
4), NKCC2 mRNA was absent from the
nephrogenic zone directly beneath the renal capsule (Fig.
1a). On
postnatal day 8, when formation of new
nephrons is completed, NKCC2 expression was present in subcapsular
tubule segments (Fig. 1a').
The first cells expressing NKCC2 were found in juxtaglomerular position of stage III nephrons. The direct contact of these cells to the vascular pole of the corresponding glomerulus suggests that they are
future macula densa cells (Fig.
2a).
With elongation of the loop past the medullary pole of the glomerulus,
the intensity of NKCC2 expression in the macula densa region increased
and extended into the ascending limb upstream to the future flow
direction (Fig. 2b). At this level,
the glomerulus was at an early capillary loop stage (Fig.
2d). Ultrastructurally, the
prospective macula densa cells already showed particular features of
the mature state such as basal and lateral membrane foldings (Fig.
2e), whereas the cells of the
immature ascending limb did not yet exhibit characteristic membrane
specializations (Fig. 2g). Apical
junctional complexes and luminal microvilli were present in cells of
the future macula densa region, even though a tubular lumen was not yet
patent (Fig. 2f). During further
maturation, the NKCC2 signal was found to extend in an upstream
direction until expression was found along the entire ascending limb
with the signal onset at the transition from the descending limb. The
point of transition was located proximal to the bend of the loop of
Henle in all newly formed immature loops. Since there is no thin
ascending limb in the immature loop, the bend itself throughout
expressed NKCC2 (Fig.
3a). At the distal end of the TAL, expression of NKCC2 consistently ended a few
cells beyond the macula densa at the site of transition to the future
DCT.
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Expression of NaPi2. To gain insight into concomitant maturation of the proximal tubule and in particular the descending limb of the developing loop, we investigated the developmental expression of NaPi2, which has been shown to be expressed along the proximal tubule in the adult nephron (10). Nephrons were showing first signs of NaPi2 when they were still located in the vicinity to the nephrogenic zone (Fig. 1b). In these stage III nephrons, NaPi2 mRNA appeared first in proximal convolutions, and signal ended at the transition to the descending limb of the primitive loop of Henle. Figure 2c shows the concomitant presence of (weak) NaPi2 mRNA signal in proximal convolutions and (stronger) NKCC2 mRNA signal in the macula densa region of the same nephron in early stage IV. In subsequent stages, NaPi2 did not occur in the descending limb until advanced NKCC2 expression in the corresponding ascending limb was found, which indicates a delayed maturation of the proximal straight tubule compared with the TAL. The complete structural maturation of the proximal nephron was achieved when NaPi2 signal had reached the renal capsule; at this time point, the entire proximal nephron was positively labeled for NaPi2 (Fig. 1b'). Whereas in the adult, NaPi2 expression showed a corticomedullary gradient with increasing signal intensity toward the medulla, such a gradient during ontogeny did not appear until day 8. In subcapsular region, however, NaPi2 expression was strong on day 8 as well (Fig. 1b').
Expression of NCC. In the mature nephron, NCC is expressed by cells along the entire DCT starting beyond the NKCC2-expressing post-macula segment of the TAL and ending at the transition into the CNT (33). A subsegmentation of the mature DCT has been described with a proximal portion (DCT1) containing exclusively NCC-expressing cells, and a distal portion (DCT2) revealing a hybrid pattern of both DCT and CNT specificities, i.e., expression of NCC as a marker of the DCT and concomitant presence of the NaCa (33) or calbindin (28) which typically label the CNT. Initial expression of NCC occurred distally in DCT segments of early stage IV nephrons; these nephrons were already located deeper within the neonatal cortex than those nephrons exhibiting first expression of NKCC2 and NaPi2 (Fig. 1c). In advanced stage IV, NCC mRNA was present along the entire DCT, beginning distally of the ascending limb of the immature loop (Fig. 3c). At the distal pole of the DCT, signal was extending into longer portions ultimately joining the CNT. At this stage, the loop of Henle presented a patent tubular lumen, whereas the epithelium of the TAL was still composed of cuboidal cells lacking interdigitation and basal striation, both typical elements of the mature state (Fig. 3d). Generally, the distal portions of the distal convolutions showed stronger NCC mRNA expression than did the proximal ones, indicating that NCC expression is initiated at the distal end of the nascent DCT. To strengthen this observation, immunolabeling of NCC was combined with NKCC2 in situ hybridization; in stage III or beginning stage IV nephrons, a portion beyond the NKCC2-expressing post-macula segment was still NCC negative (Fig. 3, b and b'). Therefore the existence of an NCC-unreactive DCT portion between the TAL and NCC-positive distal DCT portions is likely to occur during a restricted period of development. When NCC immunoreactivity (IR) was arising in the initial DCT in later stage IV, partial overlap of luminal NCC IR and NKCC2 mRNA signal was observed (Fig. 3, e and e'). By contrast, in more mature nephrons, the proximal onset of NCC IR was abrupt and showed no overlap with NKCC2. Distally, early NCC expression frequently reached the point of junction with the arcades, and double labeling with NaCa identified those portions as DCT2 segments. In more mature stages, the tubular portions properly joining the arcades were assuming a CNT-specific character with absence of NCC signal. This was particularly evident in sites where consecutive CNT generations were joining to form an arcade (Fig. 4, a and a', and c and c').
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DISCUSSION |
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Distinct transport pathways mediate salt and volume reabsorption by discrete segments of the mammalian nephron and CD (9, 13, 14, 37). The mature axial organization of transport pathways reflects a complex process of differentiation that serves both developing animal and the adult. From early in development, these epithelia adapt to function synergistically for the conservation of the bulk of glomerular ultrafiltrate (19). Yet unique developmental expression patterns of transport proteins might suggest functions that are distinct from those observed in mature nephrons. In the present experiments, we have investigated the expression of gene products involved in salt transport along the distal tubule before, during, and after the onset of filtration in the rat kidney.
Loop of Henle: TAL. Transcription of NKCC2 was observed in stage III nephrons, prior to the onset of glomerular filtration. The earliest signal was expressed in the region of the future macula densa; it then extended proximally, toward the bend of the primitive loop. Comparative analysis of the maturation of the proximal tubule indicated that NaPi2 expression began in proximal convolutions before later extending into the straight portion of the proximal tubule. NKCC2 expression into the ascending limb preceded that of NaPi2 into the descending limb. Although the two limbs of the developing loop of Henle seem to mature in parallel as judged from their morphological appearance and parallel maturation of lysosomal enzyme activities (30), the current results suggest precocious maturation of the ascending limb with regard to epithelial ion transport. In the immature loop, NKCC2 expression was detected along the entire TAL as soon as the tubular lumen had become patent. A similar spatial pattern of NKCC2 expression has been reported in the developing mouse nephron, with expression observed in the TAL in post-S-shape stages (21). The early expression of NKCC2 mRNA in the loop of Henle may not reflect functional maturity, since physiological studies have shown a low reabsorptive capacity for this segment in early postnatal stages (18). Yet, the strong NKCC2 signal at the developing macula densa and TAL of stage III nephrons does suggest that the transporter plays some functional role. The observed ultrastructural specialization of the macula densa at this stage, compared with neighboring TAL cells (see Fig. 2), would be consistent with an early functional differentiation of this epithelium, a phenomenon that has also been reported in human kidney (12).
In the adult, strong expression of NKCC2 is maintained in the macula densa (32), where it participates importantly in the luminal sensing of tubular NaCl concentration as a crucial step in tubulovascular signaling (38). It is possible that the function of the macula densa, mediation of tubuloglomerular feedback and renin secretion (38), contributes in some unknown manner to development of the juxtaglomerular apparatus or the loop of Henle. Supporting evidence for such a view comes from the observation of glomerular maldevelopment in a case of neonatal Bartter's syndrome (44). Mutations of the gene encoding NKCC2 have recently been recognized as causing some cases of the neonatal form of this disease (39). Defective expression of NKCC2 in the macula densa during development may thus also be related to the hypertrophy of the juxtaglomerular apparatus and the overexpression of renin, which typically occur in Bartter's syndrome (3, 42).
DCT. In the mature nephron, NCC expression begins abruptly at the transition from the TAL to the DCT and ends at the transition from DCT to CNT. In the developing nephron, NCC expression first appears within the distal portion of the DCT. Expression gradually extends into the post-macula segment of the TAL in late stage IV. After this stage, the transition from TAL to DCT is characterized by a short segment of NKCC2 and NCC overlap. Only later, as the nephron approaches maturity, does this overlap disappear. From the earliest stages of expression, NCC is clearly restricted to the apical cell pole, as described for the mature DCT (5, 28).
In the adult kidney, the epithelium of the segment linking midcortical and juxtamedullary DCTs to arcades has the morphological characteristics of a CNT (22). During maturation however, the epithelium of this transitional segment manifests characteristics that are intermediate between DCT and CNT patterns, at the structural level (12, 31). The current results indicate that the intermediate morphological features of the developing nephron also reflect intermediate molecular features, as well. Maturing distal convolutions that joined to form arcades frequently express both NaCa and NCC, a characteristic of the distal portion of the DCT, the DCT2, in the adult (33). During further maturation, cells that express only NaCa (not NCC) interpose between the DCT and the boundary to the arcade (see Fig. 4). Plasticity in the length of the DCT appears to be maintained in the adult kidney since in rat, an increase in the distal tubular salt load has been shown to lead to a significant increase in DNA synthesis suggesting elongation of the tubule (30).
Enhanced sodium transport capacity in the developing DCT has been postulated based on experiments documenting an inappropriate response to salt loading during ontogeny (1). This has been proposed to result from either a precocious functional maturation of the DCT or from enhanced mineralocorticoid levels that rise significantly during postnatal development (17). We have shown previously that roughly half of the DCT expresses NCC together with NaCa, rENaC, and 11HSD, whereas the other half expresses NCC but not NaCa, rENaC, and 11HSD; this agrees with functional results indicating that sodium transport by the "early" distal tubule is mediated by the thiazide-sensitive sodium-chloride cotransporter, whereas transport by the "late" distal tubule is in part electrogenic and amiloride sensitive (9, 14). Further evidence for coexpression of NCC and rENaC is derived from studies using an immortalized mouse DCT cell line. These cells demonstrate both electroneutral and electrogenic sodium transport (8).
CNT. The morphology of nascent CNTs forming arcades allows the distinction of a more mature "stem" portion comprising a major (proximal) part of the arcade and a distally adjoining "neck" portion composed of immature cells (31). NaCa IR was observed in the stem portion, whereas it was lacking in the neck region. In contrast, rENaC and 11HSD expression were expressed very early in the neck region, in the absence of NaCa. The early onset of components active in mineralocorticoid-sensitive sodium reabsorption may well have functional relevance in the CNT already during ontogeny. Supportive evidence comes from autoradiographic studies showing expression of the mineralocorticoid receptor in the maturing rabbit (15) and mouse CNT and CCD (unpublished results, N. Farman). Likewise, Kalinyak and colleagues (23) have found a threefold higher level of mineralocorticoid receptor mRNA expression in neonatal compared with adult rat. Although responsiveness of the kidney to mineralocorticoids was shown to be diminished in the postnatal rat (40), a specific effect of aldosterone on sodium reabsorption was apparent; in contrast, administration of the glucocorticoid corticosterone was without effect (40). Coupled with the current results, these results suggest that the metabolic enzyme 11HSD is already functional during development of the nephron.
The embryological origin of the CNT is not clear; some investigators have suggested that it derives from the ureteric bud, whereas others have suggested its origin is from the nephrogenic blastema (22, 31, 34). A third alternative is that it represents an epithelial hybrid, with shared properties induced by the apposition. In favor of a distal origin, the CNT contains IC and expresses rENaC and 11HSD, as does the CD. In favor of a proximal origin, however, the CNT expresses the calcium-regulating proteins NaCa and calbindin (28), which are not expressed by the CD. During development the neck zone of the arcade linking CNT and CCD expresses rENaC and 11HSD in the absence of NaCa so that it appears possible that the CNT develops from the CD by continued mitotic activity at the site of junction with the nephrogenic blastema. On the other hand, the transitional zone linking the developing CNT and DCT expresses NaCa and high levels of NCC, a protein that is characteristic of epithelia derived from nephrogenic blastema. For this reason, the current molecular data suggest that the CNT arises as a product of mutual induction from adjoining segments leading to a unique hybrid epithelium.
Ampulla and CD. In most of the mature ampullae probed for rENaC and 11HSD, the epithelium was unreactive. The developing CD, however, strongly expressed rENaC from the outermost cortical portion to the medulla. Signal for 11HSD was weak in the CCD but strong in the MCD early in development. As development progressed, 11HSD expression became equally strong in cortical and MCD, and finally became predominant in the cortex during maturity. In the immature state, the lack of 11HSD expression in outer CCD, which expresses high levels of rENaC and probably also the mineralocorticoid receptor (5, 15), suggests that glucocorticoids could act as sodium-retaining steroids during early postnatal life. In a more general sense, expression of 11HSD during fetal life has been reported from a variety of tissues, and a crucial role for this enzyme has been proposed in protecting maturing organs from elevated levels of glucocorticoids (6, 26). However, our results have shown that expression of 11HSD is spatially restricted, indicating that a general need for tissue protection via this enzyme is unlikely.
The inner MCD is known to reabsorb sodium in a mineralocorticoid- and amiloride-sensitive electrogenic manner (11, 20, 41). Duc and associates (13), however, failed to detect rENaC mRNA expression and IR in the inner MCD of mature rats. In contrast, Volk et al. (43) found rENaC activity in inner MCD cells, grown in culture. They also reported detecting all three rENaC subunits by Northern blot of rat kidney inner medulla, but the expression levels were dramatically lower than those in cortex and outer medulla. The present findings provide one explanation for these results. High level rENaC mRNA expression and IR was observed in postnatal stages on days 1, 3, 4, and 8, but it was diminished or absent at maturity. This suggests that adult rENaC expression levels are very low and may escape histochemical detection, whereas expression during development of the inner MCD is robust. The high level rENaC expression observed in cultured MCD cells may reflect a dedifferentiation of cells toward a more immature phenotype.
In conclusion, the present observations reveal a distinct pattern of cell specialization in different nephrogenic stages. NaPi2, NKCC2, NCC, NaCa, 11HSD, and rENaC are all expressed in post-S-shape stages, indicating that microanatomic differentiation of the developing nephron and CD system to some degree precedes tubular cell specialization. During further maturation, NaCl transport proteins are expressed at the early juxtaglomerular apparatus, the nascent loop, and primitive distal convolutions prior to filtrate absorption, suggesting that they play a role in mediating early cellular development, such as the regulation of gene expression, cell differentiation, or cell proliferation. The early expression of 11HSD in the maturing CNT may indicate a particular role for mineralocorticoid hormone-specific effects at the interface between the nephron and CD system. Future evaluation of targeted disruption of the described proteins could further elucidate their effects on renal morphogenesis using the rodent kidney as a model.
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ACKNOWLEDGEMENTS |
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The technical assistance of Kerstin Riskowsky and Michel Fay is gratefully acknowledged.
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FOOTNOTES |
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D. H. Ellison was supported by grants from the Department of Veterans Affairs and the American Heart Association and by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-51496-01A2. This work was performed during a tenure of an Established Investigatorship of the American Heart Association (to D. H. Ellison).
Portions of the content of this study have been presented at the 30th Annual Meeting of the American Society of Nephrology (San Antonio, TX, November 2-5, 1997).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Bachmann, AG Anatomie Charité, HU Berlin, Standort Klinikum Westend, Haus 31, Spandauer Damm 130, D-14050 Berlin, Germany (E-mail: sbachm{at}charite.de).
Received 6 August 1998; accepted in final form 13 November 1998.
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