1 Department of Physiology and Pharmacology, University of Southern Denmark-Odense University, DK-5000 Odense; and 2 Department of Cell Biology, University of Aarhus, DK-8000 Aarhus, Denmark
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ABSTRACT |
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The molecular type and localization of calcium channels along the
nephron are not well understood. In the present study, we assessed the
distribution of the recently identified 1G-subunit encoding a voltage-dependent calcium channel with T-type
characteristics. Using a RNase protection assay,
1G-mRNA
levels in kidney regions were determined as inner medulla
outer
medulla
cortex. RT-PCR analysis of microdissected rat nephron
segments revealed
1G expression in the distal convoluted
tubule (DCT), in the connecting tubule and cortical collecting duct
(CT+CCD), and inner medullary collecting duct (IMCD).
1G
mRNA was expressed in the IMCD cell line mIMCD-3. Single- and
double-labeling immunohistochemistry and confocal laser microscopy on
semithin paraffin sections of rat kidneys by using an
anti-
1G antibody demonstrated a distinct labeling at the
apical plasma membrane domains of DCT cells, CT principal cells, and
IMCD principal cells.
distal convoluted tubule; inner medullary collecting duct; voltage-operated calcium channel
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INTRODUCTION |
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TRANSMEMBRANE MOVEMENTS OF calcium are of importance for a wide range of biological processes in the kidney. Influx of extracellular calcium is required for signal transduction in renal vascular smooth muscle cells and in various tubular epithelial cells. Furthermore, the maintenance of whole body calcium homeostasis requires uptake of filtered calcium ions across the apical membranes of certain renal epithelial cells.
Voltage-gated calcium channels comprise a group of transmembrane
multisubunit proteins (3) with a central pore-forming 1-subunit containing the voltage sensor and drug
receptor. Primary distinction between classes of voltage-gated calcium
channels is between high-voltage-activated, comprising L-, N-, P/Q-,
and R-type channels, and low-voltage-activated, comprising only T-type channels (for an overview, see 16). Yu et al. (32)
demonstrated expression of at least four
1-subunit genes
in the kidney (
1A,
1C,
1D,
and
1S), all of which encode high-voltage activated calcium channels. Recently, the first gene encoding an
1-subunit with T-type characteristics,
1G, was cloned and found to be expressed in whole kidney
by Northern blotting (17, 21). We considered it relevant
to examine the nephron localization of the
1G gene product, and to this end we employed several methods. Semiquantitative estimation of the mRNA abundance in kidney regions was done by RNase
protection assay, exact localization of mRNA expression in the renal
tubules was obtained by RT-PCR analysis on microdissected tubular
segments, and, finally, the
1G-subunit protein was
localized by immunohistochemistry in semithin cryosections.
The results show that the 1G-subunit is expressed in
distinct nephron segments. We found
1G mRNA in the
distal part of the nephron as well as in the inner medullary collecting
duct. These same segments also were labeled at the apical plasma
membrane domains by using an antibody specific for
1G.
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METHODS |
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In vivo protocols. Male Sprague-Dawley rats (150-200 g), with free access to standard rodent diet (Altromin C1000, Lage, Germany) and tap water, were used for the experiments. All animal procedures conformed with the Danish law on experiments using animals as well as the guidelines for the care and handling of animals established by the US Department of Health and Human Services and published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Cell culture. The mIMCD-3 cell line (22) was obtained from the American Tissue Type Culture Collection (CRL-2123) and maintained according to instructions. In brief, the cells were grown in a 1:1 ratio of DMEM and Ham's F-12 medium, with addition of 10% fetal bovine serum. Medium was renewed two to three times weekly. Twenty hours before harvest, serum was removed. The cells were washed with PBS, and RNA was isolated by using the RNeasy kit according to manufacturer's instructions (Qiagen, KeboLab).
Microdissection of rat nephron segments.
Nephron segments were obtained by microdissection of rat kidney tissue
from a total of 10 male Sprague-Dawley rats according to the protocol
of Yang et al. (30). In brief, rats were anesthetized by
an intraperitoneal injection of mebumal, and the aorta was cannulated.
Both kidneys were perfused after the aorta had been ligated, and the
left renal vein was opened. First, kidneys were perfused with 20 ml of
cold isotonic saline followed by 20 ml of DMEM with 0.1% albumin and
collagenase A (1 mg/ml-0.2 U/mg, Roche Molecular Biochemicals). The
kidneys were removed and placed in cold saline. Thin coronal slices
were cut with a razor blade, and the slices were digested further in 25 ml DMEM with 0.1% BSA and 1 mg/ml collagenase A for 25 min at 37°C
during modest shaking. Next, the tissue was washed twice with DMEM
containing 10% FCS and kept on ice in this medium. Microdissection was
done under a stereomicroscope with sharpened forceps. The nephron
segments were identified by their localization in kidney regions and by their thickness and appearance. We obtained segments of the proximal convoluted tubule (PCT), proximal straight tubule (PST), descending thin limb of Henle's loop (DTL), thin limb of Henle's loop from inner
medulla (TL), medullary and cortical thick ascending limb of Henle's
loop (mTALH and cTALH, respectively), distal convoluted tubule (DCT),
connecting tubule and cortical collecting duct (CT+CCD), outer
medullary collecting duct (OMCD), and inner medullary collecting duct
(IMCD). To rinse off any contaminating loose cells arising from the
dissection, the isolated segments were transferred in 3-5 µl
DMEM to 500 µl fresh DMEM in a 24-well cell culture plate on ice.
Identical segments were pooled, and the total length of tubule was
assessed by using a calibrated micrometer scale built into the ocular.
The segments were transferred to 400 µl guanidinium thiocyanate (4 M)
solution, and 12 µg yeast tRNA were added as carrier. Samples were
stored at 80°C until RNA extraction. Finally, RNA was isolated by a
modified phenol-chloroform extraction protocol (6).
RT-PCR and cloning.
RT-PCR was performed as described previously (15). PCR
primers for 1G (DNA Technology, Aarhus, Denmark) were
rat, forward 5'GAA CGT GAG GCC AAG AGT '3, reverse 5'GCT TGT ATG CGT
TCC CCT '3, covering bases 3910-4130, 221 bp (GenBank accession
no. AF027984); and mouse, forward 5' CGG GAT CCT GGC AAG TCG '3,
reverse 5' CGG AAT TCG ATG ATC CGG '3, covering bases 3711-3930,
220 bp (GenBank accession no. AJ012569). Linker sequences (8 bp each)
were added to introduce EcoR I and BamH I
restriction sites for cloning.
-Actin primers were copied from Yu et
al. (32). For nephron distribution, cDNA equivalent to 1 (
1G)- or 0.2-mm tubule (actin) was used. Negative
controls included water instead of cDNA in the PCR, and carrier tRNA
with no further addition of RNA in the RT and subsequent PCR. The rat
1G amplification product was cloned in vector pSP73
(Promega) by standard methods (24). The insert was
sequenced by using SP6- and T7-specific primers. Mouse PCR products
were directly sequenced, using PCR primers.
Southern blotting.
DNA probes were synthesized with 1 µCi/µl
[-32P]dCTP (Amersham Pharmacia Biotech) and 0.05 U/µl Klenow by standard methods (24). DNA was
transferred by capillary blotting (24) to Zeta Probe GT
membrane (Bio-Rad, Copenhagen, Denmark), and hybridization of
radioactive probe was allowed overnight at 42°C (24).
The membrane was washed, and autoradiography was performed for 2-4 h.
RNase protection assays.
Animals were killed by decapitation, and organs were rapidly removed,
frozen in liquid nitrogen, and stored at 80°C. The kidneys were
separated into major regions by dissection under a stereomicroscope and
frozen in liquid nitrogen. The medullary rays were contained in the
cortical tissue, and no attempt was made to separate the outer and
inner stripe of the outer medulla. RNA was extracted according to the
acid-guanidinium-phenol-chloroform extraction protocol
(6).
Antibody against the 1G-subunit of a
voltage-dependent calcium channel.
Rabbit polyclonal antisera against rat
1G were raised by
using synthetic peptides corresponding to amino acids 1-22 of the NH2 terminal of rat
1G-subunit of a
voltage-dependent T-type calcium channel (GenBank accession no.
AF027984) with addition of a COOH-terminal cysteine. Analysis using the
BLAST program (National Library of Medicine, Bethesda, MD) revealed no
homology of the immunizing peptide with any known proteins. The
purified peptide was conjugated to keyhole limpet hemocyanin for
immunization in rabbits. For immunolocalization, the antisera were
affinity purified against immunizing peptides by using Immunobilization Kit No. 2 (Pierce, Rockford, IL).
Immunoperoxidase microscopy and laser confocal microscopy for
1G-subunit of a voltage-dependent calcium channel.
Kidneys from Munich-Wistar rats were fixed by retrograde perfusion via
the aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer
(31). Semithin paraffin sections (2-3 µm) were
incubated overnight at 4°C with anti-
1G (diluted 1:8),
and labeling was visualized with horseradish peroxidase-conjugated
secondary antibodies (P448, 1:100, DAKO, Glostrup, Denmark).
Immunolabeling controls were performed by using preabsorption of
antibodies with the
1G-immunizing peptides. As a control
on which cell types labeled positive in the distal convoluted tubule
and connecting tubule, we also labeled adjacent serial sections with
anti-
1G, anti-calbindin D-28K monoclonal mouse
antibodies (Research Diagnostics, Flanders, NJ) and polyclonal affinity-purified rabbit anti-H+-ATPase (
1-subunit)
antibodies (kind gift from Dr. Mark Knepper, NIH). For double-labeling
fluorescence microscopy, the vacuolar H+-ATPase was
localized by using a mouse monoclonal antibody (E11, diluted 1:100,
kindly supplied by Dr. S. Gluck), which was mixed with the primary
antibodies against anti-
1G (diluted 1:8). The labeling
was visualized by using both Alexa 546-conjugated goat anti-mouse
antibody and Alexa 488-conjugated goat anti-rabbit antibody (Molecular
Probes). The microscopy was carried out by using a Leica DMRE light
microscope and a Zeiss LSM510 laser confocal microscope.
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RESULTS |
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Validation of assays.
The rat 1G PCR product had the expected size of 237 bp
(Fig. 3A), and the sequence was identical to the published
rat
1G cDNA. The mouse
1G PCR product had
the expected size of 236 bp (Fig. 3B), and the sequence was
identical to the recently published mouse sequence (17),
which is 91% homologous to the rat sequence. A 32P-labeled
antisense riboprobe against the rat sequence was used to assay for the
1G-subunit mRNA in samples of total RNA from rat kidney
regions. The
1G mRNA assay was linear at least in 10- to
80-µg range whole kidney total RNA and resulted in hybrids of the
expected sizes, confirming renal expression of this subunit (not shown).
Distribution of 1G mRNA in renal regions.
RNase protection assays were performed on RNA extracted from the renal
regions (cortex, outer medulla, and inner medulla).
1G
mRNA was expressed in all regions of the kidney (Fig.
1A). The housekeeping gene
GAPDH was not differentially expressed in kidney regions (cortex
311 ± 28 cpm, n = 6; outer medulla 353 ± 50 cpm, n = 6 ; and inner medulla 226 ± 33 cpm,
n = 6, nonsignificant by ANOVA, where cpm is
counts/min). Comparisons between regions were made with
GAPDH-normalized values. Thus the
1G-subunit was 40-fold
more abundant in the inner medulla compared with cortex and outer
medulla, where levels were close to equal (Fig. 1B). By the
same method, renin mRNA was detected in the kidney cortex at levels
25-fold higher compared with outer medulla (Fig. 1B,) and in
the inner medulla no renin signal was recorded. This confirms an
accurate separation of kidney regions.
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Tissue distribution for the 1G-subunit.
It has previously been reported that
1G is predominantly
a neuronal isoform and weakly expressed in lung, kidney, and skeletal muscle (17, 21). By means of an RNase protection assay,
these tissues were compared with the kidney inner medulla for
1G expression.
1G was expressed in the
order brain > inner medulla > heart > lung
skeletal muscle (Fig. 2A).
Except for the inner medulla, which has not previously been assayed,
this is in accordance with published data (17, 21). In a
quantitative comparison of mRNA levels of brain and inner medulla (Fig.
2B), we found that inner medulla has approximately one-third
1G mRNA relative to that of brain. Comparisons of
1G mRNA levels were made by using
-actin-normalized values.
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Nephron distribution of 1G mRNA.
To determine in which segment(s) along the rat nephron
1G is expressed, we used the dual approach of RT-PCR and
immunohistochemistry. RNA was extracted from microdissected nephron
segments. By RT-PCR/Southern analysis,
1G mRNA
transcripts were consistently and abundantly detected in DCT, CT+CCD,
and IMCD. Significant levels were observed in TL, mTALH, cTALH, and
PST. Product was absent or at the limit of detection in all other
segments tested. Rat
-actin was equally amplified in all samples,
with cDNA equivalent to 0.2-mm tubule length (Fig.
3A).
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Localization of 1G-subunit in rat kidney with
immunoperoxidase microscopy and laser confocal microscopy.
For immunohistochemistry, antibodies recognizing the
NH2-terminal domain of the
1G-subunit were
raised. As demonstrated in Figs. 4 and 5, immunohistochemistry revealed
that significant labeling was associated with DCT, CT, and IMCD in rat
kidney. In contrast, glomeruli, proximal tubules, thick ascending
limbs, and CCD and OMCD were unlabeled. In the rat kidney cortex,
distinct labeling was associated with DCT and CT segments (Fig.
4, A-D). The labeling was
restricted to the very apical plasma membrane domains (arrows in Fig.
4, A-D). Immunolabeling controls using peptide-absorbed
antibodies revealed no labeling in rat kidney cortex (Fig.
4E). In kidney inner medulla, prominent labeling was
associated with IMCD, where the labeling was also confined to the
apical plasma membrane domains of principal cells (arrows, Fig.
5A). This distribution pattern
was confirmed by immunohistochemistry using anti-aquaporin-2, which
labeled principal cells in parallel semithin cryosections (not shown).
Labeling was also observed in thin limbs (Fig. 5A).
Immunolabeling control using peptide-absorbed antibodies revealed no
labeling in inner medulla (Fig. 5B).
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DISCUSSION |
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Recently, a novel 1-subunit of the voltage-gated
calcium channel family was cloned (21). The
1G-subunit shows T-type characteristics in the
expression systems tested so far; i.e., it has a low single-channel conductance and is activated at membrane potentials of
60 mV. Northern blotting has shown that
1G mRNA is expressed in
rat, human, and mouse kidney (17, 21). The purpose of the
present study was to determine the regional and nephron distribution
pattern of
1G in the rat kidney. The results confirmed
that
1G is expressed in the rat kidney and, in addition,
showed a high level of heterogeneity for
1G along the
cortical-medullary axis. Expression of
1G mRNA was
strongest in the inner medulla. RT-PCR analysis of microdissected nephron segments showed that
1G mRNA localized to the
DCT, CT and CCD, and the IMCD, whereas the OMCD was negative. The
localization of the
1G mRNA to the IMCD epithelial cells
was confirmed by detection in a mouse IMCD cell line.
Immunostaining with an antibody raised against a synthetic peptide
confirmed the presence of 1G protein in DCT, CT, and
IMCD and extended the observation to demonstrate that the labeling was
localized to the apical membrane domain of the cells. There was no
apparent immunostaining for
1G in the PST, cTAL, and CCD segments, although these cortical structures did express
1G mRNA as determined by RT-PCR. This apparent
discrepancy most likely reflects the different sensitivities of the
methods used for detection. The specificity of the reaction was
confirmed by preincubation of the antibody with the peptide used for
immunization. Immunostaining of the brain (not shown) exactly
reproduced the published localization of
1G protein
(7).
Most of the epithelial cells in the apical membranes of DCT and CT were
labeled with the 1G antibody, which colocalized with calbindin D28K immunoreactivity (Figs. 4 and 6,
A and B). This allows the conclusion that the DCT
cells and principal cells of CT express the
1G-subunit
protein. Identification of intercalated cells by an antibody against
the H+-ATPase showed scattered positive foci primarily in
the CT segment. There was no obvious colocalization with
1G in serial sections (Fig. 6, C-F), and, in
addition, the outer medullary collecting duct, which is rich in
intercalated cells, was completely unlabeled with the
1G
antibody. It should be noted that these results were obtained with
separate, but adjacent, serial sections. True colocalization with
double immunofluorescence labeling essentially confirmed these data.
Thus the
1G-subunit is exclusively expressed in the apical membrane domains of DCT cells and principal cells of the CT as
well as of the IMCD.
The only known signal for activation of T-type channels is
depolarization of the cells. Because the T-type channels are already activated at 60 mV, and because they rapidly inactivate, they are not
likely to play any functional role in nephron segments where the apical
membrane potential is more positive than this value. In late CT and
cortical collecting duct, the membrane potential is more positive
because of a dominant role of the electrogenic sodium channel (ENaC) in
epithelial transport. In contrast, DCT, early CT, and IMCD cells have
rather negative apical membrane potential (5, 28) because
both the NaCl cotransporter and ENaC are involved in NaCl absorption.
On the basis of these considerations, depolarization could
theoretically activate apical T-type channels in DCT, CT, and IMCD.
These segments are exactly the sites where we observed expression of
1G-subunits. In these segments depolarization could be
mediated by an increase in sodium absorption through ENaC, by
basolateral chloride efflux in DCT (23) and IMCD cells (4, 14), or by ischemic injury in the inner medulla,
leading to cell swelling (5) and calcium influx through
voltage-dependent calcium channels (20). Other signals may
also be relevant. The extracellular calcium receptor (CaR) is expressed
in IMCD (25) and in DCT and CT (29). Several
mechanisms are likely to be involved in the rise in intracellular
calcium, after activation of the CaR, and it is interesting to note
that activation of voltage-dependent calcium channels has been
suggested in various cell types. (8, 19).
Hormonally regulated calcium absorption is accomplished probably mainly by calcium entry via apical endothelial calcium (ECaC) channels of DCT and CT (12, 13). The ECaC is dihydropyridine insensitive. However, a number of reports exist of calcium uptake in DCT cells, which are sensitive to dihydropyridines (2, 9, 10, 18), in concentrations where they may also affect T-type channels (1, 27). Thus the presence of T-type channels in these membrane segments may provide an explanation for some of the data on apical calcium channels in DCT cells that are not explained by the ECaC channel.
In conclusion, we have provided evidence for the expression of a
low-voltage-activated calcium channel 1G-subunit in the apical membranes of DCT, CT, and IMCD cells.
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ACKNOWLEDGEMENTS |
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The authors thank Mette Fredenslund, Inger-Merete Paulsen, Zhila Nikrozi, and Mette Vistisen for expert technical assistance, and Anthony M. Carter for critical reading. We thank Dr. Henrik Hager, Dept. of Cell Biology, Univ. of Aarhus, for help on the laser scanning confocal microscopy.
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FOOTNOTES |
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Support for this study was provided by the Danish Health Science Research Council (9802855, 9601829,9503037), Ruth E. König Petersens Foundation, Novo Nordisk Foundation, Danish Heart Foundation (98-1-2-22583), Knud Øster-Jørgensens Foundation, Karen Elise Jensen Foundation, University of Southern Denmark Research Foundation, University of Aarhus Research Foundation, the University of Southern Denmark, the University of Aarhus, and the EU-Commission (EU-TMR and EU-Biotech programs).
Address for reprint requests and other correspondence: B. L. Jensen, Dept. of Physiology and Pharmacology, Winsløwparken 21.3, DK-5000 Odense C, Denmark (E-mail: bljensen{at}health.sdu.dk).
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.
Received 8 December 1999; accepted in final form 17 August 2000.
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