1 Department of Anatomy and 3 Franz Volhard Clinic, Medical Faculty of the Charité, Humboldt University, 13353 Berlin, Germany; and 2 Department of Internal Medicine, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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First published August 8, 2001; 10.1152/ajprenal.00148.2001.The
mammalian distal nephron develops a complex assembly of specialized cell types to accomplish the fine adjustment of urinary electrolyte composition. The epithelia of the distal convoluted tubule
(DCT), the connecting tubule (CNT), and the cortical collecting duct
(CCD) show an axial structural heterogeneity that has been functionally
elucidated by the localization of proteins involved in transepithelial
ion transport. We compared the distribution of the thiazide-sensitive
Na+-Cl
cotransporter (TSC), basolateral
Na+/Ca2+ exchanger (Na/Ca), cytosolic
calcium-binding proteins calbindin D28K and parvalbumin,
and the key enzyme for selective aldosterone actions,
11
-hydroxysteroid-dehydrogenase 2 (11HSD2), in the distal convolutions of the mouse. In the mouse, as opposed to the rat, we
found no clear subsegmentation of the DCT into a proximal (DCT1) and a
distal (DCT2) portion. The TSC was expressed along the entire DCT. Na/Ca and calbindin D28K were similarly expressed
along most of the DCT, with minor exceptions in the initial portion of
the DCT. Both were also present in the CNT. Parvalbumin was found in
the entire DCT, with an occasional absence from short end portions of
the DCT, and was not present in CNT. 11HSD2 was predominantly located
in the CNT and CCD. Short end portions of DCT only occasionally showed
the 11HSD2 signal. We also observed an overlap of 11HSD2 immunoreactivity and mRNA staining. Our observations will have implications in understanding the physiological effects of gene disruption and targeting experiments in the mouse.
distal convoluted tubule; connecting tubule; sodium-calcium
exchanger; calbindin; parvalbumin; 11-hydroxysteroid-dehydrogenase
type 2
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INTRODUCTION |
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MAMMALIAN KIDNEYS ARE
CAPABLE of adapting urinary sodium and chloride excretion
according to intake across a wide range of salt intakes with high
precision. The fine adjustments take place in the distal nephron.
Successive portions of the renal tubule are formed to adapt to this
function, and an axial heterogeneity of the distal segments has been
defined (11, 13). In the cortex, the thick ascending limb
of Henle's loop (TAL), the distal convoluted tubule (DCT), the
connecting tubule (CNT), and the collecting duct (CCD) have been
identified. The specific transport properties of these epithelia are
accomplished by the expression of proteins representing cotransporters,
exchangers, and ion channels. These proteins govern ion movement from
one side of the cell to the other. The distribution, ontogeny, and
functional aspects of these proteins in the mammalian distal nephron
have been reviewed elsewhere (2, 23). In the rat, the
electroneutral cation-chloride cotransporter (Na+-K+-2Cl) has been localized
to the TAL (11). The thiazide-sensitive Na+- Cl
cotransporter (TSC; also termed
NCC) has been localized to the more proximal (DCT1) and distal
(DCT2) DCT segments (18). The epithelial sodium
channel (ENaC), the mineralocorticoid receptor, and the enzyme
11
-hydroxysteroid-dehydrogenase type 2 (11HSD2), the key enzyme for
selective aldosterone actions in the distal nephron (14,
25), have been localized to the terminal portion of the DCT, the
CNT, and the collecting duct of rats and mice (6, 7, 24).
Colocalization studies with antibodies against proteins involved in
distal tubular calcium metabolism have permitted a detailed definition
of these portions in rat and other species (3-5, 16,
18-20). Mutations in all of the sodium transport-related proteins have been associated with human disease, including
hypertension, hypotension, acid-base derangements, and other elements
of disturbed distal fluid and electrolyte regulation (23,
26). The rat has been used as a universal model; however, gene
disruption and targeting strategies have only been successful in the
mouse. Recently, sophisticated physiological techniques have been
adapted to the mouse. Nevertheless, the precise anatomic
localization of these proteins in the mouse is imperfectly defined.
Therefore, we analyzed the localization of the TSC and 11HSD2 along the
precise distal tubular segments of the mouse, as defined by the
colocalization of proteins involved in calcium metabolism. To establish
the segmentation of the mouse nephron in this respect, we defined the
distribution of the Na+/Ca2+ exchanger (Na/Ca),
calbindin D28K, and parvalbumin in agreement with published
data from the rat and mouse nephron (3, 5, 7, 13, 18, 24).
Additional evidence came from comparative microanatomic analysis and
histochemical double-staining with a TAL marker.
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METHODS |
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Experiments were performed in adult male NMRI mice (body wt 25-30 g; Schönwalde, Berlin, Germany). The protocol was conducted after due approval according to American Physiological Society guidelines. Animals were allowed free access to water and standard chow. The animals were killed under Nembutal anesthesia. The kidneys were perfused retrograde through the abdominal aorta using PBS adjusted to 330 mosmol/kgH2O with sucrose, pH 7.4, for 20 s as described earlier (24). Next, 3% paraformaldehyde in PBS was infused for 5 min, followed by the first solution for an additional minute. The kidneys were removed and cut into 3- to 5-mm-thick slices and shock-frozen in liquid nitrogen-cooled isopentane.
Immunohistochemistry. To analyze distal segments and segmental portions, we used well-characterized, segment-specific antibodies as follows: rabbit polyclonal antibody against a fusion protein containing the entire NH2-terminal tail of the mouse TSC (Ref. 6; 1:100 dilution in PBS containing 5% skim milk); guinea pig polyclonal antiserum against a fusion protein from rabbit corresponding to the basolateral Na/Ca (1:50 dilution; Ref. 20); mouse monoclonal antibody (ascites fluid) against calbindin D28K (1:500 dilution; Sigma, Deisenhofen, Germany); rabbit polyclonal antiserum against a fusion protein corresponding to human 11HSD2 (Ref. 14; 1:200 dilution); goat polyclonal antiserum against parvalbumin (1:5,000 dilution; Swant, Bellinzona, Switzerland); and rabbit polyclonal antiserum against Tamm-Horsfall protein (gift of J. Hoyer, Philadelphia, PA; 1:200 dilution).
Immunolabeling was performed on cryostat sections of 5 µm thickness. After blocking with 5% skim milk-PBS, pH 7.4, sections were incubated with primary antibody for 2 h at room temperature and then at 4°C overnight. When double labeling was performed, the antibodies were administered consecutively. Thorough rinsing in PBS was followed by signal detection with Cy3-conjugated goat anti-rabbit IgG serum (1:250 dilution in skim milk-PBS), Cy2-conjugated goat anti-guinea pig (1:60 dilution), or donkey anti-mouse IgG (1:100 dilution) for 1 h at room temperature (Dianova, Hamburg, Germany). In double-labeling experiments, suitable secondary antibodies coupled to different fluorochromes were applied. Double labeling with primary antibodies raised in the same host, in the case of TSC and 11HSD2, was performed, with each antibody applied to one of two consecutive sections. Cell nuclei were simultaneously stained, in part, with 4',6-diamino-2-phenylindole (DAPI; Sigma) diluted 1:100 in PBS. For ultrastructural preembedding histochemistry and immunoperoxidase labeling, an established protocol was applied (1); for incubation of 20-µm-thick slices generated in a vibratome, anti-11HSD2 antibody was used at dilutions between 1:50 and 1:200. Sections were incubated overnight in microtiter plates. After epon embedding, ultrathin sections were cut and viewed in the electron microscope. Controls for immunohistochemistry were done by replacing primary antibodies with skim milk-PBS controls.In situ hybridization. An 11HSD2 riboprobe was prepared from a respective 248-bp cDNA fragment of rat kidney 11HSD2. The fragment was subcloned into the EcoRV site of a Bluescript KS+ vector (Stratagene, La Jolla, CA) as described elsewhere (6). Riboprobes were synthesized and labeled by in vitro transcription using digoxigenin-labeled UTP and T3 or T7 RNA polymerase (Boehringer Mannheim, Mannheim, Germany) to generate sense-control or antisense transcripts, respectively. In situ hybridization of 5- to 7-µm cryostat sections was done as described elsewhere (18). Riboprobes were used at concentrations between 10 and 15 ng/ml, and hybridization was performed at 40°C. Combined in situ hybridization and immunohistochemistry was done on the same tissue section with the primary antibody applied in parallel with the anti-digoxigenin alkaline phosphatase conjugate. The antibody was marked with the Cy3-labeled secondary antibody before detection of the riboprobe (24).
Ultrastructural analysis. Ultrathin sections were viewed with a Leo electron microscope. Light microscopy sections were viewed with a Leica DMRB light microscope equipped with interference contrast optics and an HBO fluorescence lamp. Light microscopic images were obtained with a digital camera (Spot 32, Diagnostic Instruments, Munich, Germany) and processed with Meta View 3.6a software (Universal Imaging, West Chester, PA).
Quantitative estimation of cell numbers. Cells in the regions of TAL-DCT and DCT-CNT junctions were quantitatively evaluated, using combined staining of DAPI for the cell nuclei counts and combined staining with specific antibodies to the epitopes of interest. For all quantifications, at least three sections each from a minimum of two different animals were used.
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RESULTS |
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In agreement with published nomenclature (13), the distal convolutions of the mouse kidney take their origin at the TAL-DCT junction, where the typically high DCT epithelium abruptly begins after a short postmacula segment of the TAL. This transition was additionally verified by the concomitant end of TAL-specific immunolabeling by antibody against Tamm-Horsfall protein (not shown). As in the rat, mouse DCT is proportionally well represented (18). It shows a few intercalated cells in its terminal portion and then continues into the equally well-developed CNT segment. CNT profiles have numerous intercalated cells, their epithelium is lower than in DCT, and they are typically located near the interlobular vasculature.
The anti-TSC antibody showed an apical immunoreactive signal in the DCT of the mouse nephron. The onset of TSC localization in this segment was verified by establishing the TAL-DCT junctions, as well as the transitions from DCT to CNT. The terminal DCT portions were further identified by the occurrence of one to three TSC-unreactive intercalated cells per longitudinally sectioned DCT-CNT junction. Identity of these junctions was additionally verified by their vicinity to the interlobular vessels.
Figure 1 shows inter- and intrasegmental
heterogeneity of the distal convolutions with respect to
immunohistochemical distribution of the basolateral Na/Ca. Generally,
Na/Ca and TSC are colocalized in the DCT; Na/Ca immunoreactivity starts
with the onset of the DCT and extends farther, beyond the end of the
TSC signal and into the CNT. The entire CNT is Na/Ca positive; the CCD
is negative. Within the DCT, Na/Ca showed marked differences in
intensity. A significant initial DCT portion (usually >20
cells/sectioned profile) had a low signal intensity. The remaining
distal portions of the DCT showed strong Na/Ca labeling intensity that
was equal to that in CNT. Single, scattered cells of distal DCT
portions showed a particularly strong Na/Ca immunofluorescence (see
Fig. 4A).
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We next studied the heterogeneity of the distal convolutions with
respect to the immunohistochemical distribution of calbindin (Fig.
2). Generally, calbindin and TSC were
colocalized in the DCT; calbindin immunoreactivity began either sharply
at the TAL-DCT junction, or within an initial portion of the DCT, and
extended farther, beyond the end of the TSC signal and into the CNT.
The entire CNT and the CCD were strongly calbindin positive. All
intercalated cells were negative. In the initial portion of the DCT,
the calbindin signal was absent from the first one to two cells per
longitudinally sectioned TAL-DCT junction in 8% of all cases evaluated
(see Fig. 4B) and weak in 22%. In 70% of all cases, a
calbindin signal was present from the very beginning of the DCT.
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Figure 3 shows the distribution of
parvalbumin in the distal segments. A weak parvalbumin signal was
occasionally found in terminal TAL portions of varying length,
including parts of the macula densa. With the onset of the DCT, the
parvalbumin signal consistently assumed maximal strength that was
maintained throughout almost the entire length of the DCT. Only a short
terminal DCT portion, measuring an average length of 2.7 cells in
longitudinal sections of DCT-CNT junctions, was lacking parvalbumin
immunoreactivity in less than half of all cases (Fig.
4C).
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We next studied the distribution of 11HSD2 in the distal segments.
Figure 5 shows that 11HSD2
immunoreactivity and mRNA signals were principally colocalized in the
CNT and CCD (Fig. 5, C and D), whereas the
majority of the DCT profiles were entirely free of a 11HSD2 signal.
Only occasionally, a terminal short portion of the DCT, roughly
corresponding in length to the parvalbumin-negative terminal DCT
portions, showed concomitant expression of immunoreactive 11HSD2 and
TSC, as revealed in consecutive sections (Fig. 5, A and
B). Intercalated cells were 11HSD2 negative. To strengthen these observations, the presence of immunoreactive 11HSD2 in terminal DCT cells and in CNT cells could also be confirmed at the
ultrastructural level by preembedding immunoperoxidase staining (Fig.
6). Here, the signal was mainly localized
in the subapical cytosol and, to a lesser extent, also in the basal
cell compartments between the mitochondria and the basal labyrinth,
respectively. A comprehensive overview of these results is presented in
Fig. 7.
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DISCUSSION |
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Our study defines the localization of specific proteins involved in electrolyte handling along the mouse DCT and the neighboring segmental portions of up- and downstream positions, i.e., in TAL and CNT. The DCT, as defined by its unique property to express the TSC, revealed a rather homogenous phenotype with respect to the distribution of the other gene products analyzed. The products did not exhibit the same expression patterns that formerly led us to define the DCT1 and DCT2 subsegments in the rat distal nephron. TAL-DCT and DCT-CNT junctions in mice were characterized by relatively sharp transitions. Accordingly, initial and terminal DCT portions only occasionally displayed short zones, a few cells in length, which did or did not express segment-specific gene products. These irregular patterns indicated an internephron heterogeneity, although to a modest extent.
Expression of the Na/Ca was localized in the entire DCT. This pattern is in contrast to that in the rat, which lacks a Na/Ca signal in DCT1 (2, 18). The colocalization of Na/Ca and TSC in the entire DCT of the mouse suggests a more important functional relationship between the two gene products than in the rat. Diuretics affecting the DCT also stimulate calcium transport across the basolateral membrane into the interstitium of the rat (8, 29), and the molecular candidates for an apical calcium entry pathway in the DCT have been identified (4). On the other hand, in the rabbit, Na/Ca and apical calcium channel expression are absent in the DCT and begin only with the CNT, so that presence of the TSC and 1,25(OH)2D3-regulated calcium reabsorption are mutually exclusive in this species (3, 4).
Double staining of calbindin and TSC also showed major differences between the rat and mouse. In the rat, overall calbindin expression was weak. Only in the transition segments at the DCT-CNT junctions was a calbindin signal revealed that was as great as the calbindin signal in the CNT (16, 19). However, in contrast to that in the rat, the abundance of calbindin in mouse DCT was considerably higher along the entire DCT, with the occasional exception of a short initial portion of this segment.
Strong expression of gene products involved in mouse distal tubular calcium handling further agrees with the significant expression of parvalbumin in the entire DCT, with the occasional exception of a terminal DCT portion. The irregular presence of parvalbumin in TAL agrees with previous work done in the rat (5). The association between parvalbumin and calbindin in our study was relatively similar to that described in the rat, supporting the notion that the proteins are associated in the membrane transport process.
Mineralocorticoids have a well-established effect on renal Na+ and K+ transport in the distal tubule. The typical target cells are the principal cells of the collecting duct (9, 10). However, further evidence suggests that sodium transport may also be regulated by mineralocorticoid hormones in other nephron segments. Aldosterone acts via nuclear hormone receptors that function as transcription factors regulating the expression of various genes (29). Recent studies have shown that aldosterone stimulates the TSC in rat kidney (27). Accordingly, both the mineralocorticoid receptor and the metabolic enzyme isoform 11HSD2 have been identified in the DCT2 and in the CNT (6, 14, 24, 28). 11HSD2 plays an important physiological role in maintaining mineralocorticoid specificity by metabolizing cortisol to cortisone, which has no affinity for the mineralocorticoid receptor and thus exerts a prereceptor barrier function in the typical mineralocorticoid target cells (25). Accordingly, DCT segments expressing the TSC actively metabolize corticosterone via 11HSD2 when dissected from rabbit kidney (28). These findings are further in line with the observation that 11HSD2-deficient mice display the phenotype of apparent mineralocorticoid syndrome and are hypertensive (12).
Labeling with probes for 11HSD2 expectedly showed strong staining of CNT cells and CCD principal cells. The signal was pertinent until the transition to the inner medulla. The overall cytosolic localization of the enzyme, as revealed by ultrastructural labeling, agreed with its biochemical role within the cell. However, in the DCT, only an occasional 11HSD2 signal was shown. Thus we found no support for the notion that the DCT in the mouse is a major target for mineralocorticoid action.
We previously described the axial distribution of 11HSD2 in DCT2, CNT, and CCD, but not in DCT1 and TAL, of rat kidney (6, 24). In addition, we showed a coincident expression of the amiloride-sensitive ENaC that underlines the mineralocorticoid sensitivity of these segments, because the activity of this channel is related to aldosterone levels (for review, see Refs. 23 and 29). A similar localization of ENaC was also later confirmed in the mouse (17). A preliminary report on selective malformation of an initial part of the DCT in TSC null-mutant mice also claimed axial heterogeneity of the mouse DCT (15). These results are not supported by our findings. The reason for the discrepancy cannot be clarified at present. However, ENaC localization in the late DCT of the mouse was very weak compared with in the CNT. This finding favors the interpretation that aldosterone-dependent sodium handling in this portion of the nephron is not of major importance.
Another implication of our anatomic findings is a possible relationship between sodium and calcium homeostasis involving mineralocorticoid-related effects. The parallel localization of the Na/Ca and 11HSD2 expression in mouse CNT suggests a mineralocorticoid hormone-dependent component of calcium transport in this segment, although this assumption awaits experimental consolidation. In this regard, it is known that the aldosterone antagonist spironolactone reduces renal calcium excretion (22). Furthermore, in states of mineralocorticoid excess such as Conn's syndrome, calcium wasting has been described (21).
In summary, our findings show that the DCT in mice presents an almost
complete colocalization of gene products involved in thiazide-sensitive
Na+-Cl cotransport and in calcium handling,
along with only minor expression of 11HSD2. In contrast to DCT of rat
kidney, in mouse kidney DCT appears to lack a clear subsegmentation
into DCT1 and DCT2. The concentrated colocalization of gene products
involved in calcium handling with the TSC suggests that a functional
relationship between Na+-Cl
cotransport and
Ca2+ handling may exist in this segment. The
relative absence of 11HSD2 from the DCT and its presence in CNT and CCD
will have implications regarding steroid effects in mice; it remains to
be experimentally verified whether targets of mineralocorticoid action
are located downstream of the DCT. Our observations will aid in an
understanding of the physiological effects produced by gene disruption
and targeting experiments in the mouse.
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ACKNOWLEDGEMENTS |
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We appreciate the technical help of Kerstin Riskowsky. We thank W. B. Reeves, Department of Internal Medicine, University of Arkansas, Little Rock, AR, for the generous gift of an 11HSD2 antibody and Dr. B. Kaissling for advising us to include parvalbumin immunostaining in our analysis.
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FOOTNOTES |
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This work was supported by grants-in-aid from the Deutsche Forschungsgemeinschaft (to S. Bachmann).
Address for reprint requests and other correspondence: S. Bachmann, AG Anatomie/Elektronenmikroskopie, Charité CVK, BMFZ, Augustenburger Platz1, 13353 Berlin, Germany (E-mail: sbachm{at}charite.de).
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. Section 1734 solely to indicate this fact.
First published August 8, 2001;10.1152/ajprenal.00148.2001
Received 11 May 2001; accepted in final form 16 August 2001.
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