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*
Department of Cell Physiology, University Medical Center Nijmegen,
Nijmegen, The Netherlands
Department of Biochemistry, University Medical Center Nijmegen, Nijmegen,
The Netherlands
Department of Endocrinology, University Medical Center Nijmegen, Nijmegen,
The Netherlands
Department of Pharmacology, Jichi Medical School, Tochigi,
Japan.
Correspondence to Dr. René Bindels, Department of Cell Physiology, University Medical Center Nijmegen, Institute of Cellular Signalling, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: +31-24-3614211; Fax: +31-20-8675224; E-mail: reneb{at}sci.kun.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase, a renal enzyme that
controls the synthesis of the biologic active form of vitamin D, calcitriol,
or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3),
and for the vitamin D receptor
(1,2).
1,25(OH)2D3 is of primary importance because it enhances
the active Ca2+ absorption in small intestine and stimulates
Ca2+ reabsorption in kidney
(3,4,5).
The distal nephron determines the final excretion of Ca2+ into the
urine and is a target for regulation of the extracellular Ca2+
concentration. Ca2+ reabsorption in these latter segments is active
and generally envisioned as a three-step process consisting of passive entry
of Ca2+ across the apical membrane, cytosolic diffusion of
Ca2+ bound to calbindin-D28K, and active extrusion of
Ca2+ across the opposite basolateral membrane by the Na+
-Ca2+ exchanger and the plasma Ca2+ ATPase
(4,6).
A major breakthrough was the recent discovery of the Ca2+ influx
protein, named epithelial Ca2+ channel (ECaC)
(7). ECaC is present primarily
in 1,25(OH)2D3-responsive epithelia and exhibits the
distinctive properties for being the gatekeeper of active Ca2+
reabsorption in the distal part of the nephron
(6,8). In the present study, we postulated that 1,25(OH)2D3 acts on the distal part of the renal tubule by increasing the expression of ECaC, which will result in increased Ca2+ reabsorption. In general, the genomic mechanism of 1,25(OH)2D3 action involves direct interaction of the vitamin D receptor with regulatory domains on the promoter region of the gene known as vitamin D—responsive elements (VDRE). The VDRE has been reported to consist of two imperfect repeats separated by three nucleotide pairs (9). On the basis of promoter sequence analysis in vitamin D—responsive genes such as calbindin-D28K (10), calbindin-D9K (11), osteocalcin (12), and the 24-hydroxylase gene (13,14), several VDRE were identified.
To provide evidence for this hypothesis, we raised vitamin D—deficient rats to study the effect of 1,25(OH)2D3 on the expression of ECaC. In addition, the human ECaC promoter was elucidated and analyzed for VDRE. Using ECaC-specific ribonuclease protection and immunofluorescence assays, we could demonstrate that decreases in circulating levels of 1,25(OH)2D3 resulted in a marked decline in ECaC mRNA and protein expression. In agreement with this finding, the human ECaC promoter sequence was shown to contain several putative VDRE. Taken together, this study indicates that the recently elucidated ECaC, which is present exclusively in the distal part of distal convoluted tubule (DCT) and connecting tubule (CNT), is an important target for 1,25(OH)2D3 to regulate renal Ca2+ excretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum Parameters
Total Ca2+ concentrations in serum were measured with the use of
a colorimetric assay kit as described previously
(16). The concentration of
1,25(OH)2D3 in serum was assessed with the use of a
radio-receptor assay after extraction of the samples followed by paper
chromatography, as described previously
(17).
Ribonuclease Protection Assay
Riboprobes for rat ECaC (nucleotides 2130 to 2330)
(7), calbindin-D28K
(nucleotides 1420 to 1570)
(18), and ß-actin
(nucleotides 2220 to 2420)
(19) were prepared by
transcription of subcloned cDNA fragments into pGEM-Teasy (Promega, Madison,
WI) with the use of either SP6 or T7 RNA polymerases and
[
-32P]UTP (3000 Ci/mmol; Amersham, Buckinghamshire, UK) as
described previously. The ribonuclease protection assay (RPA III kit; Ambion,
Austin, TX) was performed with the use of total RNA (5 to 20 µg), isolated
from kidney cortex with Trizol reagent (Life Technologies BRL, Breda, The
Netherlands). This RNA was hybridized with labeled riboprobe (105
cpm) for 16 h at 42°C and treated with RNase A/T1 (25/100 U/ml)
mix for 1 h at 37°C. The protected fragments were precipitated, heat
denatured, and separated on 6% (wt/vol) denaturing polyacrylamide gels. The
gels were exposed to a film (Kodak, Rochester, NY) for quantification of
radioactive signals under conditions in which linearity was achieved. The mRNA
expression levels were quantified by computer-assisted densitometry with the
use of a flatbed scanner in combination with Molecular Analist software
(Imaging Densitometer GS-690; Biorad, Richmond, CA).
Immunoblotting
Kidney cortex tissue was removed, immediately frozen in liquid nitrogen,
and homogenized in phosphate-buffered saline. All samples (20 µg of
protein) were separated on 12% (wt/vol) sodium dodecyl
sulfate—polyacrylamide gel electrophoresis gels and blotted to
polyvinyldifluoride membranes (Immobilon-P; Millipore Corporation, Bedford,
MA). Blots were incubated for 16 h with calbindin-D28K antibody
(1:10000), and immunoreactive protein was detected with the use of the
enhanced chemiluminescence method as described previously
(5). The protein expression
levels were quantified by computer-assisted densitometry with the use of a
flatbed scanner as described above.
Immunohistochemistry
Kidney tissue was cut into pieces, placed in 1% (wt/vol)
periodate-lysine-paraformaldehyde fixative for 2 h at room temperature, and
incubated overnight at 4°C in phosphate-buffered saline containing 15%
(wt/vol) sucrose as described previously
(20). Subsequently, kidney
samples were frozen in liquid nitrogen and 7-µm frozen sections were cut
for different staining procedures. The kidney sections were stained with
guinea pig anti-ECaC antiserum (1:200) as described previously. Sections that
were double stained for ECaC and thiazide-sensitive NaCl co-transporter (NCCT)
were incubated simultaneously for 16 h at 4°C with antiserum against ECaC
and NCCT (1:3200) (20). Double
staining of ECaC with calbindin-D28K was performed simultaneously
with anti-ECaC antiserum (1:200) and rabbit antiserum against
calbindin-D28K (1:200) as described previously. To visualize ECaC,
calbindin-D28K and NCCT, we stained sections with goat anti-guinea
pig-Alexa 594-conjugated anti-IgG (1:300), goat anti-rabbit-Alexa
488-conjugated anti IgG (1:300), or goat anti-mouse Alexa 488-conjugated
anti-IgG (1:300), respectively (Molecular Probes, Eugene, OR). All negative
controls, including sections incubated with preimmune serum, antiserum
preabsorbed for 1 h with 10 µg/ml
ECaC—glutathione-S-transferase fusion protein, or conjugated
antibodies alone, were devoid of any staining. Photographs were taken with a
Zeiss Axioskop microscope (Thornwood, NY) equipped for epifluorescence
illumination with the use of Kodak EPH P1600X film.
5' Rapid Amplification of cDNA Ends and Isolation of ECaC
Genomic Fragments
The 5' terminus of human ECaC (hECaC) cDNA was identified with the
use of the 5' rapid amplification of cDNA ends (RACE) technique on
adapter-ligated human cDNA (Clontech, Palo Alto, CA) as described by
Müller et al.
(21). An EcaC-specific
antisense primer (5'-GGCAAGCAGTGCGCGCACCAGGTTCA-3') at nucleotide
position 506 to 480 on cDNA and an adapter-1 sense primer
(5'-CCATCCTAATACGACTCACTATAGGGC-3') were used for 5' RACE.
Subsequently, a 5' radiolabeled hECaC-specific genomic DNA probe,
containing the 5'UTR and exon 1, was used to screen a genomic library
(Stratagene, La Jolla, CA) to obtain the promoter region of hECaC. After
several rounds of screening, one specific clone that contained a part of the
5' ECaC gene including the promoter region could be identified. Dye
termination sequencing was performed with the use of the BigDye Termination
kit (Perkin Elmer, Foster City, CA) following the manufacturer's protocol, and
separation was carried out at the Research Sequence Facility, University
Medical Center Nijmegen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of 1,25(OH)2D3 on ECaC Expression in
Kidney
To investigate the effect of 1,25(OH)2D3 on ECaC
expression in the kidney, we performed RNase protection assays. Total RNA from
kidney cortex was isolated from vitamin D—deficient and —repleted
rats and control rats fed a normal standard diet and hybridized with an
ECaC-specific radioactive labeled RNA probe
(Figure 1). These data show
that 48 h of treatment of vitamin D-deficient rats with
1,25(OH)2D3 resulted in a marked increase in ECaC mRNA
abundance (183 ± 9%), comparable with the expression level observed in
control rats. To confirm the correct size of the protected ECaC fragment, we
used 1 µg of rabbit ECaC cRNA transcribed from pSPORT-ECaC (Promega,
Madison, WI) (7). As a control,
ß-actin—specific hybridizations were performed with an equal
quantity of total RNA (Figure
1). Scanning of the ß-actin corresponding bands revealed
little or no variation in the densities, thus excluding the possibility that
the difference in ECaC expression between vitamin D—deficient and
—repleted rats was due to unequal loading. In the next experiment, the
abundance of ECaC protein was examined.
Figure 2 shows representative
immunofluorescence labeling of distal tubules in sections of kidney cortex
from control rats and vitamin D—deficient and —repleted rats.
Reduction of the 1,25(OH)2D3 levels had a major effect
on ECaC protein expression as indicated by the virtually complete absence of
staining in the kidney cortex, whereas normalization of the
1,25(OH)2D3 levels in the repleted animals restored ECaC
expression to levels comparable to those observed in control rats
(Figure 2, A through C). ECaC
is localized predominantly to the apical membrane of distal tubule segments in
vitamin D—repleted and control animals. Thus, the increased expression
in the renal cortex observed in the RNase protection assays is not associated
with a major redistribution of ECaC within the distal tubule segments. The
ECaC antibody used in this study has been characterized extensively but is,
unfortunately, not working on immunoblots
(20). To semiquantify ECaC
protein expression, we counted immunopositive tubules in 10 random microscopic
fields for each animal. Figure
2D depicts the average values obtained from four vitamin
D—deficient rats and demonstrates that ECaC expression is virtually
abolished. This is in contrast to the vitamin D—repleted rats in which
immunopositive tubules were observed easily for each animal and in number
identical to those in normal-fed rats
(Figure 2, E and F).
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Effect of 1,25(OH)2D3 on
Calbindin-D28K Expression in Kidney
The expression level of the vitamin D—dependent calcium binding
protein, calbindin-D28K, was determined at the mRNA and protein
levels. RNase protection assays clearly demonstrated an upregulation of
calbindin-D28K mRNA in vitamin D—repleted (286 ± 21%;
P < 0.005, n = 4) compared with vitamin D—deficient
animals (Figure 3A). The
immunoblot shown in Figure 3B
clearly demonstrates an increase in calbindin-D28K after repletion
of 1,25(OH)2D3 (312 ± 47%; P < 0.005,
n = 4). Immunohistochemistry confirmed this upregulation of
calbindin-D28K protein by 1,25(OH)2D3
treatment of the deficient rats (Figure 3,
C and D). The observed protein expression levels for
calbindin-D28K in the repleted rats were not significantly
different from control rats that were fed a normal standard diet (data not
shown).
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Distribution of ECaC in Rat Kidney Cortex
Co-localization studies of ECaC, calbindin-D28K, and the
thiazide-sensitive NCCT were performed to investigate the distribution of
these proteins in rat kidney. Figure
4A shows distal tubule segments expressing ECaC in a control rat.
As shown before, ECaC was located predominantly to the apical membrane.
Co-localization studies revealed that ECaC and calbindin-D28K were
bracketed exclusively to the same tubule segments
(Figure 4B). Within the
ECaC-immunopositive tubules, some negative cells (indicated by white
arrowheads) were observed, which were identified as intercalated cells.
Figure 4, C and D, shows double
labeling of rat kidney cortex for the presence of ECaC and NCCT. Some distal
tubule segments (indicated by an asterisk) stained intensively for NCCT but
were negative for ECaC. Importantly, a transition (arrowheads) was found from
NCCT immunopositive to negative within one tubule, whereas the whole segment
stained for ECaC. Because the NCCT-positive segments were identified
previously as the first part of DCT (DCT1)
(22), this finding suggests
that ECaC and, therefore, calbindin-D28K are present exclusively in
the more distal part of DCT (DCT2) and the CNT
(Figure 4E). There was no
indication that ECaC expression in the deficient rats was decreased
selectively in DCT2 or CNT.
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Identification of the Human ECaC Promoter
To identify the transcription initiation site, we performed 5'-RACE
experiments on hECaC cDNA. Figure
5 shows this transcription site, designated +1, at 90 bp upstream
from the translation initiation ATG codon. Subsequently, a human genomic
library was screened with the use of a 5' genomic hECaC probe containing
the 5'UTR, exon 1, and the 5' part of the adjacent intron,
resulting in the isolation of a single clone containing a part of the 5'
ECaC gene including the transcription initiation site and 2.4-kb upstream
information (Figure 5) (GenBank
AJ278604). Neither a classical TATA box nor a CCAAT box was identified close
to the 5' region of the transcription initiation site. However, at
position -25, the sequence ATAAA was located. Although TATA and CCAAT boxes
were positioned farther upstream, their distance to the transcription site is
considered too far to have a promoter function. The hECaC promoter contains a
number of potential cis-acting elements recognized by
well-characterized transcription factors that may play a role in the
regulation of the ECaC gene (Figure
5). Importantly, several putative VDRE were identified in the ECaC
promoter region (indicated by black boxes). In addition, many other regulatory
sites were identified, including sites for activator protein 1, 2, and 3
(AP-1, AP-2, and AP-3), a stimulatory protein (SP-1) box, and a NF
B
recognition domain. Evaluation of the physiologic meaning of these sites in
the gene awaits further studies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The vitamin D—deficient status of the rats used in our study was illustrated by severe hypocalcemia. Administration of 1,25(OH)2D3 completely normalized this situation within 48 h and was accompanied by a modest upregulation of ECaC mRNA and a marked increase in ECaC protein expression. These observations suggest that in addition to an increase in mRNA abundance, translational regulation of ECaC may take place, which results in increased channel activity at the apical cell surface. In theory, the enhanced ECaC mRNA expression could be due to increased transcriptional activity and/or mRNA stabilization. The steroid character of 1,25(OH)2D3 and the presence of VDRE in the ECaC promoter, however, are in line with a regulation at the transcriptional level, but extensive promoter analysis is necessary to demonstrate the functional role of these elements.
In addition, 1,25(OH)2D3 enhances the renal expression of calbindin-D28K as demonstrated in previous studies (reviewed in reference 23) and by our group in primary cultures of CNT cells (5). Because the activity of ECaC is tightly controlled by the cytosolic Ca2+ concentration, it is essential that the number of ECaC channels at the plasma membrane be matched by the cytosolic Ca2+ buffering capacity of these cells (24). The present observation that both ECaC and calbindin-D28K are synchronically upregulated demonstrates that this requirement is fulfilled. The necessity of sufficient Ca2+ buffering for optimal ECaC functioning is underlined by the conspicuous co-localization of ECaC and calbindins in Ca2+ transporting tissues (21).
Importantly, the hECaC promoter contains several putative VDRE within an area ranging between 20 and 2000 bases upstream, which might be involved in the observed 1,25(OH)2D3-dependent ECaC mRNA expression. The core binding motif sequences of the VDRE known to date are rather divergent, but the hexameric sequence RRKNSA (R = A or G, K = G or T, S = C or G) seems to be a distinct consensus sequence (25). In human and rat osteocalcin promoters, an AP-1 consensus sequence closely juxtaposed to the VDRE has been identified. It has been demonstrated that AP-1 sites synergistically enhance the activation by 1,25(OH)2D3 (13). In the ECaC promoter, putative VDRE are surrounded by an AP-1 site. However, further promoter analysis will be required to identify the key regulatory sites involved in vitamin D—mediated transcription. The proximal promoter region does not contain typical TATA or CCAAT boxes; however, a similar site is present at -20 (ATAAA). Although these elements have been demonstrated to play an important role in the transcription machinery, many genes have been described without these sequences (26). Alternatively, SP-1 regions (GGGCGG) present at position -209 may drive transcription as indicated for other genes. For the chicken VDR promoter, which contains no TATA box but possesses GC boxes or SP-1 sites, a series of deletion promoter constructs established that the proximal GC boxes are the major drivers of gene transcription (27).
Micropuncture studies of superficial nephron segments of the rat have demonstrated that the distal part of the nephron is responsible for reabsorption of 15% of filtered Ca2+. The portion of the distal tubule normally assessed by micropuncture is a combination of the DCT, CNT, and initial collecting tubule. However, the exact contribution of the individual segments to active Ca2+ transport was still unresolved (4,6,8,22). Immunolocalization studies in rabbit kidney cortex demonstrated that the expression of ECaC is restricted to the connecting tubule that is the predominant Ca2+ transporting segment in this species (4,6,8). In other species, including rat, Ca2+ reabsorption seems to be located primarily in DCT (4,22). However, the present localization study demonstrates that like in rabbit, there is very little overlap in the localization of NCCT and ECaC in rat kidney. The expression of NCCT is limited to DCT cells. In human, rat, and mouse, expression of this co-transporter extends from the proximal end of the DCT into a transitional segment, referred to as DCT2, that shares properties of both DCT and CNT (22). Conversely, ECaC and calbindin-D28K are restricted to DCT2 and CNT. Thus, thiazide-sensitive NaCl cotransport and 1,25(OH)2D3-regulated Ca2+ reabsorption are spatially dissociated in that NCCT is restricted to DCT1 and ECaC is restricted to CNT, whereas DCT2 is a transitional segment that expresses both transport systems. This functional separation has important implications for understanding the hypocalciuria observed in patients with Gitelman's syndrome and during administration of thiazide diuretics (28,29). So far, the effects of thiazides on Ca2+ reabsorption were explained by a hyperpolarization that increased the driving force for transcellular Ca2+ transport (4,29). Because the localization of NCCT and ECaC are dissociated, other mechanisms must be at work.
The regulation of ECaC by 1,25(OH)2D3 may shed new
light on Ca2+ metabolism under pathophysiologic circumstances. The
treatment with vitamin D that has been proved to be beneficial in various
clinical situations (e.g., the prevention of rickets during infancy)
now can be explained at the molecular level. Primary or secondary involvement
of ECaC can be expected in several pathologic situations. Among these,
conditions associated with hypercalciuria certainly are of interest because of
the dominant localization of ECaC in the kidney. Closely correlated with
hypercalciuria is nephrolithiasis, which, because of its high prevalence, has
a considerable socioeconomic impact. Intensive investigations, therefore, have
been performed in the past to clarify the underlying mechanisms
(30,31).
Recent studies suggest that the pathogenesis is heterogeneous, because
different molecular pathways have been causally related to this disease
(32). Interest of research
centers around defective regulation of Ca2+ homeostasis and the
related vitamin D metabolism. Potential candidate genes have been screened for
their involvement but several genes (e.g., 1-hydroxylase) have
been excluded (33).
Alternatively, dysregulation of 1,25(OH)2D3-sensitive
Ca2+ reabsorption through ECaC that controls partly the
Ca2+ flux into the blood compartment could be one factor in the
pathogenesis of Ca2+-related kidney stone disease.
After the recent molecular identification and subsequent electrophysiologic characterization of ECaC, the present study demonstrates unequivocally that the expression of ECaC in DCT2 and CNT is controlled by the calciotropic hormone 1,25(OH)2D3. Future studies may substantiate the role of 1,25(OH)2D3-responsive ECaC expression in the pathogenesis of Ca2+ homeostasis-related disorders.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase gene in patients with
pseudovitamin D-deficiency rickets. N Engl J Med338
: 653-661,1998,25-dihydroxyvitamin D3 24-hydroxylase gene promoter and
identification of two vitamin D-responsive elements. Biochim
Biophys Acta 1263:1
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regulates ATP-dependent calcium transport in basolateral plasma membranes of
rat enterocytes. Biochim Biophys Acta689
: 170-172,1992
-hydroxylase
locus is not linked to calcium stone formation or calciuric phenotypes in
French-Canadian families. J Am Soc Nephrol9
: 425-432,1998[Abstract]
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C.-T. Lee, S. Shang, L.-W. Lai, K.-C. Yong, and Y.-H. H. Lien Effect of thiazide on renal gene expression of apical calcium channels and calbindins Am J Physiol Renal Physiol, December 1, 2004; 287(6): F1164 - F1170. [Abstract] [Full Text] [PDF]
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G. Mossetti, D. Rendina, R. Viceconti, G. Manno, V. Guadagno, P. Strazzullo, and V. Nunziata The relationship of 3' vitamin D receptor haplotypes to urinary supersaturation of calcium oxalate salts and to age at onset and familial prevalence of nephrolithiasis Nephrol. Dial. Transplant., September 1, 2004; 19(9): 2259 - 2265. [Abstract] [Full Text] [PDF]
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J. Loffing, V. Vallon, D. Loffing-Cueni, F. Aregger, K. Richter, L. Pietri, M. Bloch-Faure, J. G.J. Hoenderop, G. E. Shull, P. Meneton, et al. Altered Renal Distal Tubule Structure and Renal Na+ and Ca2+ Handling in a Mouse Model for Gitelman's Syndrome J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2276 - 2288. [Abstract] [Full Text] [PDF]
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H. F. Cantiello Regulation of calcium signaling by polycystin-2 Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1012 - F1029. [Abstract] [Full Text] [PDF]
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T. Nijenhuis, J. G.J. Hoenderop, and R. J.M. Bindels Downregulation of Ca2+ and Mg2+ Transport Proteins in the Kidney Explains Tacrolimus (FK506)-Induced Hypercalciuria and Hypomagnesemia J. Am. Soc. Nephrol., March 1, 2004; 15(3): 549 - 557. [Abstract] [Full Text] [PDF]
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R. J. W. Diepens, E. den Dekker, M. Bens, A. F. Weidema, A. Vandewalle, R. J. M. Bindels, and J. G. J. Hoenderop Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport Am J Physiol Renal Physiol, March 1, 2004; 286(3): F483 - F489. [Abstract] [Full Text]
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S. N. Kip and E. E. Strehler Vitamin D3 upregulates plasma membrane Ca2+-ATPase expression and potentiates apico-basal Ca2+ flux in MDCK cells Am J Physiol Renal Physiol, February 1, 2004; 286(2): F363 - F369. [Abstract] [Full Text] [PDF]
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M. Bodding, C. Fecher-Trost, and V |
