1Division of Pediatric Nephrology, Department of Pediatrics Mount Sinai School of Medicine, New York 10029; 2Division of Nephrology and Hypertension, Department of Medicine, Winthrop University Hospital, Mineola, New York 11501; and 3Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
Submitted 19 May 2003 ; accepted in final form 13 June 2003
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ABSTRACT |
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maxi-K channel; iberiotoxin; in vitro microperfusion; intracellular calcium concentration; mechanoregulation; development
Urinary K excretion is derived almost entirely from K secretion in the
connecting tubule and cortical collecting duct (CCD)
(13). In contrast to the high
rates of K secretion observed in CCDs isolated from adult animals and
microperfused in vitro at physiological flow rates, segments isolated from
neonatal animals show no significant net K transport until after week
3 of postnatal life (38).
Of note is that the rate of net Na absorption in the CCD at 2 wk of age is
60% of that measured in the adult
(38). By 6 wk of age, the rate
of net K secretion in CCDs perfused at a flow rate of
1 nl ·
min1 · mm1 is
comparable to that observed in the adult
(38). These results indicate
that the low rates of urinary K excretion characteristic of the newborn kidney
are due, at least in part, to a low secretory capacity of the CCD.
The CCD is comprised of two cell populations. Principal cells reabsorb Na and secrete K (24, 37, 47), whereas intercalated cells are thought to primarily function in acid-base homeostasis but can reabsorb K in response to dietary K restriction or metabolic acidosis (44, 46, 63). Within the principal cell, Na reabsorbed across apical epithelial Na channels (ENaCs) is extruded by the basolateral Na-K-ATPase in exchange for the uptake of K. Cell K diffuses down a favorable electrochemical gradient, established by electrogenic Na absorption, into the tubular lumen through apical K-selective channels. Thus K secretion in the CCD requires Na absorption and the presence of conducting apical K-selective channels.
Two types of apical K-selective channels have been functionally identified in the rabbit principal cell by patch-clamp analysis (41, 42). The prevalence of the low-conductance secretory K (SK) channel and its high open probability at the resting membrane potential (12, 42, 57) led to the premise that this channel mediates K secretion under baseline conditions. The high-conductance (>100 pS) K channel, rarely open at physiological membrane potentials, exhibits kinetics similar to those of the maxi-K channel described by others (18, 22, 35). In contrast to the SK channel, detected within the CCD solely in principal cells, high-conductance maxi-K channels, activated by membrane depolarization, stretch, elevation of intracellular calcium concentration ([Ca2+]i), or hypoosmotic stress (11, 14, 21, 22, 35, 48, 50, 51), are present in both principal and intercalated cells of the CCD (35). In fact, the density of maxi-K channels in intercalated cells exceeds that detected in principal cells (35).
An increase in tubular fluid flow rate in the fully differentiated CCD
stimulates net K secretion (9,
15,
16,
28,
40). We recently reported that
flow-stimulated net K secretion is associated with increases in net Na
absorption, presumably due to stimulation of ENaC activity
(43), and increases in
[Ca2+]i in both principal and intercalated
cells (58,
59). On the basis of the
sensitivity of the response to TEA and charybdotoxin
(58) and the
electrophysiological evidence for apical SK and maxi-K channels in the rabbit
CCD (41,
42), we proposed that
flow-dependent K secretion is mediated by the maxi-K channel, as had been
suggested by others (13,
35,
50). In contrast to the robust
increase in net K secretion elicited by flow in the adult rabbit CCD,
flow-dependent net K secretion cannot be elicited until after week 4
of postnatal life (38). Yet,
patch-clamp analysis reveals high-conductance (110 ± 6 pS) K channels,
activated by membrane depolarization, in 10% of cell-attached patches of
the apical membranes of rabbit principal cells at
2 wk of age
(41).
The purpose of the present study was to examine the cellular and molecular
basis for the delayed postnatal appearance of flow-stimulated K secretion in
the CCD, first observed 2 wk after baseline K secretion is established.
Given our previous detection of high-conductance K channels in the neonatal
rabbit principal cell by patch-clamp analysis, we initially speculated that
the absence of flow-stimulated K secretion early in life was due to a limited
capacity for flow to augment Na absorption and/or induce a rise in
[Ca2+]i. However, once these flow-induced
responses were shown not to be limiting, we used RT-PCR, Southern blotting,
and indirect immunofluorescence to show that expression of maxi-K channel mRNA
and protein is developmentally regulated.
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MATERIALS AND METHODS |
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Isolation of single tubules. The kidneys were removed via a midline incision and sliced into 2-mm coronal sections, and single tubules were dissected freehand in cold (4°C) Ringer solution containing (in mM) 135 NaCl, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 lactate, 6.0 L-alanine, 5.0 HEPES, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O, as previously described (59). A single tubule was studied from each animal.
For in vitro microperfusion studies, each isolated tubule was immediately transferred to a temperature- and O2-CO2-controlled specimen chamber, assembled with a no. 1 glass coverslip (VWR Scientific, Media, PA) as its base. The CCD was mounted on concentric glass pipettes and cannulated. For measurement of [Ca2+]i, CCDs were affixed, basolateral membrane down, to coverslips previously painted with a 1-µl drop of Cell-Tak (Collaborative Biomedical Products, Bedford, MA), as previously described (59). Each tubule was perfused and bathed at 37°C with Burg's perfusate containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 Na lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (59). During the 60-min equilibration period and thereafter, the perfusion chamber was continuously suffused with a gas mixture of 95% O2-5% CO2 to maintain pH of the Burg's solution at 7.4 at 37°C. The bathing solution was continuously exchanged at a rate of 10 ml/h using a peristaltic syringe pump (Razel, Stamford, CT).
For molecular studies, single tubules were dissected in cold PBS containing 10 mM vanadyl ribonucleoside complex (Sigma, St. Louis, MO) to inhibit RNA degradation, as previously described (3). On average, 1518 mm total of CCDs or proximal tubules were pooled for each tubule sample. Tubules were rinsed three times in cold 1x PBS and transferred to a 1.5-ml microcentrifuge tube for immediate RNA extraction. Dissection time was limited to 2 h to ensure RNA integrity.
Measurement of cation fluxes. Isolated CCDs were microperfused in
vitro as previously described
(58). Transport measurements
were performed in the absence of transepithelial osmotic gradients; thus water
transport was assumed to be zero. Samples of tubular fluid were collected
under water-saturated light mineral oil by timed filling of a precalibrated
30-nl volumetric constriction pipette. The flow rate was varied from
1 to 6 nl · min1 ·
mm1 by adjusting the height of the perfusate
reservoir.
For cation transport rates, three to four samples of tubular fluid were collected at each flow rate and analyzed for K and Na concentrations. The K and Na concentrations of perfusate and collected tubular fluid were determined by helium glow photometry and the rates of net cation transport (in pmol · min1 · mm1 tubular length) were calculated, as previously described (38). To determine the concentration of K and Na delivered to the tubular lumen, ouabain (100 µM) was added to the bath at the conclusion of each experiment to inhibit all active transport, and three to four samples of tubular fluid were then obtained for analysis similar to that described above. The calculated ion fluxes were averaged to obtain a single mean rate of ion transport for the CCD at each flow rate. The sequence of flow rates was randomized within each group of tubules to minimize any bias induced by time-dependent changes in ion transport.
The transepithelial voltage (Vte) was measured between symmetrical calomel electrodes continuous with the perfusion pipette via a 0.16 M NaCl agarose bridge and referenced to the bath, as previously described (38). Voltages were monitored using a high-impedance electrometer (World Precision Instruments, New Haven, CT). Readings were taken at the midpoint of each timed collection and averaged.
A subset of CCDs, as indicated, was perfused with the scorpion venom toxin iberiotoxin (99% purity; Sigma), a selective inhibitor of the high-conductance Ca2+-activated maxi-K channel (5, 52). The final concentration of 50 nM was prepared by diluting the toxin in the luminal perfusate.
Measurement of intracellular Ca2+ concentration. After equilibration, microperfused tubules were loaded with 20 µM of the acetoxymethyl ester of fura-2 (Calbiochem, La Jolla, CA) added to the bath for 20 min. With the use of a Nikon Eclipse TE300 inverted epifluorescence microscope linked to a cooled Pentamax CCD camera (Princeton Instruments) interfaced with a digital imaging system (MetaFluor, Universal Imaging, Westchester, PA), intracellular Ca2+ concentration ([Ca2+]i) was measured in individually identified fura-2-loaded cells visualized using a Nikon S Fluor x40 objective (numerical aperture 0.9, WD 0.3), as previously described (59). We previously showed that fura-2-loaded intercalated cells, identified by their selective apical binding of rhodamine PNA, appear more brightly fluorescent under epifluorescence illumination compared with principal cells, although steady-state [Ca2+]i does not differ between the two cell populations (59). Autofluorescence was not detectable at the camera gains used.
CCDs were alternately excited at 340 and 380 nm and images, acquired every 2 to 10 s, were digitized for subsequent analysis. At the conclusion of each experiment, an intracellular calibration was performed, using 10 µM EGTA-AM in a Ca2+-free bath and then a 2-mM Ca2+ bath containing ionomycin (10 µM), as previously described (59). Standard equations were used to calculate experimental values of [Ca2+]i (17). Two to four principal and intercalated cells were analyzed in each CCD.
RT-PCR and Southern blotting. RNA was extracted from tubules using a modified acid guanidinium-phenol-chloroform RNA extraction method (6). Briefly, each sample of tubules was placed in 50 µl of extraction buffer containing guanidinium thiocyanate and allowed to sit on ice for 5 min. Five microliters of 2 M Na acetate, 50 µl of water-saturated phenol, and 20 µl of chloroform-isoamyl alcohol were added to the buffer solution containing the tubules and gently mixed. The sample was then placed on ice for 15 min and centrifuged at 14,000 rpm for 10 min at 4°C. The aqueous layer was precipitated with 100 µl of ice-cold 100% ethanol. To this, 0.6 µl of the coprecipitant linear acrylamide (Ambion, Austin, TX) were added and precipitated for 1 h at 20°C. Segment-specific RNA was recovered by centrifugation (30 min at 4°C) at 14,000 rpm. The pellet was resuspended in 5 µl of diethyl pyrocarbonate-treated water. With the use of the standard protocol provided by Invitrogen (Carlsbad, CA), the extracted RNA was treated with RNase-free DNase I and incubated at room temperature for 15 min. EDTA (25 mM) was added to inactivate the enzyme before heating to 65°C.
Reverse transcription was performed using oligo dT under standard
techniques with Superscript II (Invitrogen). Amplification of the slo
pore region was conducted using a DNA Thermal Cycler 480 (Perkin Elmer,
Norwalk, CT), using sequence-specific probes (sense:
5'-GTTACGGGGACGTTTATGC-3'; antisense:
5'-CCAACTTCAGCTCTGCAAG-3'), 1.5 mM MgCl2, and Platinum
Taq DNA Polymerase (Invitrogen), predicted to generate a product size
of 608 bp. In total, 40 cycles of denaturation (94°C, 1 min),
annealing (58°C, 1 min), and extension (72°C, 1 min) were conducted.
Amplification of GAPDH was performed using a sense primer
(5'-GCTGAACGGGAAACTCACTG-3') and an antisense primer
(5'-TCCACCACCCTGTTGCTGTA-3'), expected to yield a product size of
307 bp. The PCR products were size-fractionated by electrophoresis on a
2% agarose gel and visualized by UV fluorescence after ethidium bromide
staining to verify that the PCR products were of expected size. The sequences
of the PCR products were verified by direct sequencing (ABI Prism model 3700
Sequencer).
In some cases, reverse transcriptase was omitted from the reaction as a negative control for amplification of genomic DNA. RNA extracted from whole kidney of adult rabbits was used as a positive control. Data from tubule samples were omitted from further analysis if the sample failed to generate a GAPDH amplification product.
RT-PCR of single tubules was followed by Southern blotting with a labeled probe (MegaPrime DNA Labeling System, Amersham, UK) encoding the slo pore region, generated using the primer sets identified above applied to RNA extracted from whole kidney of adult rabbit. PCR products were depurinated with 0.25 N HCl for 8 min. Alkaline denaturation of the gel was then performed twice with a solution containing 1.5 M NaCl and 0.5 M NaOH for 15 min, followed by neutralization with 1 M Tris and 1.5 M NaCl, pH 7.4, twice for 15 min. With the use of a standard transfer technique with 10x SSC buffer, the DNA was blotted onto a Hybond-N nylon membrane (Amersham). After overnight transfer, the membrane was baked for2hinan80°C vacuum oven and prehybridized at 42°C in Ultrahyb solution (Ambion) for 1 h. The labeled probe was then added and the membrane was incubated overnight at 42°C. The blot was washed twice with 2x SSC and 0.1% SDS at 42°C for 5 min. Visualization was performed with a phosphorimager (Molecular Dynamics).
Western blotting of maxi-K channel protein in kidney. Rabbit
kidneys were homogenized in a buffer containing 250 mM sucrose and 10 mM
triethanolamine, pH 7.6, supplemented with protease inhibitors (1:100 dilution
of Protease Inhibitor Cocktail Set III, Calbiochem, San Diego, CA). Aliquots
of the protein lysate (100 µg) were subject to 315% SDS-PAGE and
transferred to an Immobilon-NC membrane (Millipore, Waltham, MA). Blots were
blocked overnight with 5% nonfat dried milk in PBS (8 mM Na phosphate, 2 mM K
phosphate, 140 mM NaCl, 10 mM KCl, pH 7.4) plus 0.05% Tween 20 (PBS-Tween),
probed with an affinity-purified antibody raised in chicken against the
sequence CTANRPNRPKSRESRDKQN corresponding to a COOH-terminal region of mouse
maxi-K -subunit (Aves Labs) at a concentration of 0.5 µg/ml in
PBS-Tween 20 for 3 h at room temperature. Alternatively, blots were probed
with the anti-maxi-K antibody that was preincubated overnight at 4°C with
the peptide immunogen at a concentration of 20 µg/ml. Bound antibody was
detected following incubation with a 1:2,500 dilution of horseradish
peroxidase-conjugated goat anti-chicken IgG (Kirkegaard and Perry
Laboratories, Gaithersburg, MD) by chemiluminescence (Western Blot
Chemiluminescence Reagent Plus, New England Nuclear, Boston, MA).
Immunofluorescence localization of maxi-K channels in the kidney. Coronal sections of rabbit kidneys were fixed in 4% paraformaldehyde and sucrose and embedded in medium (Cryo-Gel, Instrumedics). Serial 4-µm-thick sections were cut on a cryostat (Leica CM1900) and collected on Superfrost microscopic slides (Fisher Scientific). Sections were hydrated and washed with PBS three times and quenched with PBS-0.02% Gly-1% BSA (PBS buffer solution). The tissue was then permeabilized with PBS buffer solution and 0.1% Triton X-100 for 10 min, blocked with PBS buffer solution for 30 min, and then with 5% milk with 0.05% Tween 20 in PBS buffer solution for 60 min.
Tissue sections were incubated with the anti-maxi-K antibody (6 µg/ml) for 1 h at room temperature. For peptide competition experiments, peptide was added to the primary antibody at a concentration of 80 µg/ml. After incubation with primary antibody, the sections were washed three times for 5 min with PBS buffer solution with 0.05% Tween 20. The secondary antibody, a Cy3-conjugated Affinipure F(ab')2 fragment donkey anti-chicken IgY, was applied at a 1:3,000 dilution (stock concentration of 1.5 mg/ml) in PBS buffer with 0.05% Tween 20 for 45 min at room temperature; each section was colabeled with FITC-conjugated dolichos biflorus agglutinin (DBA; 5 µg/ml), a principal cell marker (64). Sections were then washed three times with PBS buffer with 0.05% Tween 20 for 5 min followed by three quick washes with PBS solution. All sections were mounted on coverslips with phenylene diamine mounting media.
Confocal microscopy was performed on a Leica TCS SL equipped with krypton and green and red helium-neon lasers. Images were acquired with the use of a x100 plan-apochromat objective (numerical aperture 1.4) and appropriate filter combination [Kalman filter (n = 4)]. Images were saved in a tag-information-file format, and the contrast levels of the images were adjusted in the Photoshop program (Adobe, Mountain View, CA) on Power PC G-4 Macintosh (Apple, Cupertino, CA). The contrast-corrected images were imported into Freehand (Macromedia, San Francisco, CA) for viewing.
Statistics. All results are expressed as means ± SE;
n equals the number of animal or tubule samples used for in vitro
microperfusion studies and RT-PCR. Multiple (3) blots and amplifications
were performed with different tubules or RNA samples that were isolated from
different animals. CCDs from young animals were harvested from at least five
litters of animals. Comparisons were made by paired and unpaired
t-tests as appropriate. Significance was asserted if P <
0.05.
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RESULTS |
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Effect of age on flow-stimulated net Na absorption. To determine
whether the absence of flow-stimulated K secretion in the 4-wk-old CCD was due
to a limited capacity of the tubule to augment net Na absorption with an
increase in flow rate, net Na absorption was measured in the same tubules as
described directly above. As shown in Fig.
1B, net Na absorption was similar in the three age groups
studied at a flow rate of 1 nl · min1
· mm1. After an increase in tubular flow
to
6 nl · min1 ·
mm1, net Na absorption (in pmol ·
min1 · mm1)
increased approximately fourfold (4 wk: 27.9 ± 2.1 to 106.8 ±
4.1; 5 wk: 26.2 ± 1.3 to 90.8 ± 5.4; 6 wk: 31.3 ± 2.2 to
105.9 ± 10.2) in each age group studied (P < 0.05),
suggesting that the mechanisms mediating flow-dependent Na absorption are
established before the onset of flow-stimulated K secretion. There was no
significant difference (P = not significant) noted between the rates
of net Na transport at the
6-nl ·
min1 · mm1
flow rate among the three age groups studied.
Effect of iberiotoxin on flow-stimulated net K secretion. To
confirm that flow-stimulated net K secretion is mediated by a high-conductance
maxi-K channel, the effect of the specific channel inhibitor iberiotoxin was
tested in three CCDs isolated from rabbits 6 wk of life. In the absence of
iberiotoxin, net K secretion increased from 7.8 ± 2.1 to 23.0 ±
1.1 pmol · min1 ·
mm1 (P < 0.05) as the tubular flow
rate was increased from 1.4 ± 0.1 to 6.4 ± 0.2 nl ·
min1 · mm1
(Fig. 2A). The
flow-stimulated component of net K secretion was inhibited by addition of 50
nM iberiotoxin to the luminal perfusate during a period of sustained high flow
(6.9 ± 0.4
pmol·min1·mm1,
P < 0.01 compared with transport rate in absence of iberiotoxin)
(Fig. 2A). Luminal
iberiotoxin did not affect flow-stimulated net Na absorption, which averaged
101.8 ± 6.6
pmol·min1·mm1
in these same tubules (P = not significant compared with transport
rate in absence of iberotoxin) (Fig.
2B).
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Flow-induced [Ca2+]i transients. We previously reported that an increase in tubular flow rate in the adult rabbit CCD is associated with an increase in [Ca2+]i (59). To determine whether flow induces comparable increases in [Ca2+]i in maturing CCDs, CCDs from 1- to 5-wk-old rabbits were loaded with fura-2 and [Ca2+]i was measured. Baseline [Ca2+]i was lower in principal cells at 1 wk of age (53.4 ± 14.3 nM, P < 0.05) compared with older age groups (97.8 ± 26.9, 115.8 ± 42.2, 99.1 ± 14.0, and 77.1 ± 15.6 nM at 2, 3, 4, and 5 wk, respectively, n = 3 in all groups except for 5-wk-old subjects, where n = 4) (Fig. 3). Resting [Ca2+]i in intercalated cells in 1-wk-old CCDs (70.4 ± 11.5 nM) was not significantly different from that measured at 2 (86.6 ± 26.0 nM) and 3 (125.2 ± 43.8 nM) wk of age, but it was less than that detected in the older age groups (100.7 ± 4.7 and 120.7 ± 13.3 nM at 4 and 5 wk, respectively) (Fig. 3). An acute increase in tubular fluid flow rate, sufficient to increase tubular diameter by 25.3 ± 3.4%, increased [Ca2+]i in both principal and intercalated cells at all ages (Fig. 3). However, the peak [Ca2+]i elicited by high flow was significantly lower in 1-compared with 5-wk-old principal (P < 0.01) and intercalated (P < 0.03) cells.
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Developmental expression of slo message in single tubules. To examine the developmental expression of slo mRNA in the CCD, RT-PCR was performed on single CCDs (n = 8 to 20 samples/age group) and proximal tubules (n = 7 to 11 samples/age group) using primers directed against the pore region. Slo transcripts were not consistently detected until week 4 of life in the CCD (Figs. 4 and 5). CCD samples were split for selective amplification of slo and GAPDH; a single band of appropriate size for GAPDH was detected at all age groups (Fig. 4), indicating that the absence of slo transcripts early in life was not due to RNA degradation. In the absence of reverse transcriptase, bands were not detected. The sequence of the amplified product showed a 99.5% homology to the published rabbit slo channel at both the nucleotide and predicted amino acid level.
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Southern blotting of the RT-PCR gels, using a channel pore region-specific
probe, revealed a signal in only one of four CCD samples harvested from
2-wk-old kidneys (Figs. 4 and
5). Transcripts were never
detected in any CCD samples isolated from 1- and 3-wk-old rabbits
(Fig. 5), whereas all CCDs
dissected from animals 4 wk of life showed slo transcripts.
Slo transcripts were also not detected in any proximal tubule segment
at any age, although all tubule samples expressed GAPDH
(Fig. 6).
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Western analysis of maxi-K channel protein in kidney homogenate.
An immunoblot of whole rabbit kidney lysate was performed to characterize the
anti-maxi-K channel antibody. Polypeptides with apparent molecular weights of
200 and
90 kDa were recognized by the antibody but not by antibody
preincubated with the immunizing peptide
(Fig. 7). The 90-kDa
polypeptide has a lower apparent molecular mass than was reported for the
rabbit
-subunit isoform rbSlo1 expressed in HEK293 cells (146 kDa)
(56) and may represent a
splice variant of rbSlo1. The 200-kDa polypeptide may represent an
-subunit dimer.
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Immunolocalization of maxi-K channels in the kidney. Cryosections
of adult rabbit kidney were labeled with antibodies directed against the
-subunit of the maxi-K channel. Principal cells were labeled with DBA.
Labeling with both markers was observed in discrete groups of tubules,
presumably collecting ducts (Fig.
8). Apical localization of maxi-K
-subunits in DBA-positive
tubules was observed in intercalated (i.e., DBA negative) cells
(Fig. 8, arrows). Intracellular
localization of maxi-K channels in CCDs was observed in both DBA-positive
principal (Fig. 8, arrowheads)
as well as DBA-negative intercalated cells. These results suggest that apical
membrane expression of maxi-K channels is restricted mainly to intercalated
cells and are in agreement with the previous electrophysiological finding that
the density of conducting maxi-K channels in CCDs is greater in intercalated
than principal cells (35).
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We also examined the expression of maxi-K channels in the developing kidney
of the rabbit. No significant immunostaining with the anti-maxi-K antibody was
observed until week 5 of postnatal life, at which time staining was
observed in both DBA-negative intercalated cells as well as DBA-positive
principal cells (Fig. 9). As in
adults, apical membrane expression of maxi-K channels was limited to
DBA-negative cells (Fig. 9,
arrows), and intracellular staining was observed in both DBA-positive
(Fig. 9, arrowheads) and
DBA-negative cells (Fig. 9).
These results correlate well with developmental appearance of flow-dependent K
secretion and with message encoding the maxi-K -subunit.
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DISCUSSION |
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We recently proposed that flow-stimulated K secretion is mediated by the Ca2+/stretch-activated high-conductance maxi-K channel (58) previously identified by electrophysiological analysis of the apical membrane of rat and rabbit CCDs (35, 41). This notion is supported by the present observation that iberiotoxin, a scorpion venom toxin known to block the maxi-K channel with high affinity (5, 52), prevents flow-stimulated K secretion without affecting baseline K secretion (Fig. 2A).
Two subunits of the maxi-K channel have been cloned
(2,
8,
23): a pore-forming
-subunit, a member of the slo family of K channels originally
cloned from Drosophila, and a regulatory
-subunit. Coexpression
of both subunits in Xenopus laevis oocytes enhances the voltage,
Ca2+, and charybdotoxin sensitivity of the channel
compared with expression of the
-subunit alone
(20,
30). All slo genes
generate multiple transcripts via alternative splicing
(1,
4,
25,
33). Heterologous expression
of the unique variants reveals differences in their voltage,
Ca2+, and hormonal sensitivity
(54,
61) and, as suggested in more
recent studies, their subcellular localization and association with
interacting proteins (32).
Two alternatively spliced transcripts of the maxi-K channel -subunit
have been cloned from the rabbit medullary thick ascending limb (MTAL), a
segment that expresses conducting channels
(33). Rbslo1 contains
a 174-bp insert immediately following the predicted S8 transmembrane domain
(33). Although the amino acid
sequence of rbslo1 is highly homologous to slo channels
cloned from mouse, human, and chicken
(33), the rabbit subunit
exhibits enhanced Ca2+ and voltage sensitivities
compared with the other cloned isoforms due to the COOH-terminus insertion
sequence (19). Rbslo2
has a 104-bp deletion between the S9 and S10 regions
(33). Rbslo1 and
2 are expressed in glomeruli, thin and thick limbs of Henle, and
cortical and medullary collecting ducts, but they are rarely detected in
proximal tubule (33).
Functional studies provide evidence for the differential expression of
rbslo transcripts in unique segments and cell types in the rabbit
kidney. For example, stretch activation (application of pipette suction) of
maxi-K channels in cell-attached or inside-out patches on rabbit CCD
intercalated cells persists even after chelation of free
Ca2+ with EGTA in the pipette or the bath solutions,
implying that stretch activation of these channels is not mediated by
increased Ca2+ entry into the cell and is not
Ca2+ sensitive
(35). In contrast, the rabbit
connecting tubule maxi-K channel is not stretch activated in the absence of
ambient Ca2+
(50).
We considered it theoretically possible that the differential expression of
unique slo splice variants during maturation could account, in part,
for our early (2 wk after birth) electrophysiological detection of
high-conductance K channels, activated by depolarization
(41), in 10% of principal
cells, and relatively late appearance (5 wk of age;
Fig. 1A) of
flow-stimulated K secretion in the rabbit CCD. Given that our primer pairs
were designed to amplify the highly conserved pore region of the
-subunit common to all splice variants, our failure to detect any
-subunit-specific transcripts in CCDs isolated from animals <4 wk
(Figs. 4 and
5) likely reflects either that
1) levels of expression are below the threshold for detection by our
molecular analyses or 2) high-conductance K channels detected in the
neonatal principal cell are encoded by a gene distinct from slo. It
should be stated that the high-conductance K channels were not rigorously
characterized in the neonatal CCD but were assumed to be maxi-K channels based
on their conductance and similarity in kinetics to the well-described rat
channel (35). Future efforts
should be directed at a thorough electrophysiological analysis of these
channels in the maturing CCD.
Functional evidence for high-conductance, Ca2+-activated K channels has been demonstrated not only in the MTAL, connecting tubule, and collecting duct, but also in proximal tubule (31, 33, 52). However, neither we (Fig. 6) nor others (33) identified slo transcripts in proximal tubules of mammalian kidneys, raising uncertainty to the molecular identity of the proximal tubule channel. The functional maxi-K channel detected in this segment may be encoded by another gene.
An unexpected finding of the present investigation was the
immunolocalization of the -subunit of the maxi-K channel predominantly
to the apical surface of intercalated cells. Whereas principal cells also
exhibited anti-maxi-K channel antibody labeling, the signal appeared to be
localized primarily within the cell, with little apical label detected. These
results, although in good agreement with the previous electrophysiological
findings of a higher density of maxi-K channels in intercalated than principal
cells (35), suggest that the
flow-activated component of net K secretion may be mediated by intercalated
cells, a cell population not traditionally considered to participate in net K
secretion. Of note is that these cells possess an apical H-K-ATPase
(7,
45). Others
(45,
60) have suggested the
presence of an apical K channel in intercalated cells that allows for K that
is absorbed via the H-K-ATPase to recycle back into the tubular fluid, to
ensure a continuous supply of luminal K for the apical pump. Perhaps this
function is accomplished by the maxi-K channel.
Our finding that a rapid increase in tubular flow rate leads to an increase in both principal and intercalated cell [Ca2+]i in the differentiating CCD is consistent with our recent observations in the fully differentiated CCD (26, 59). Emerging evidence suggests that the apical cilium present in principal but not intercalated cells is a flow sensor (34, 36). We recently proposed that flow-induced shear or hydrodynamic impulses at the cilium or apical membrane of the fully differentiated CCD stimulate release of Ca2+ from inositol-1,4,5-trisphosphate (IP3)-sensitive internal stores and influx from the extracellular space (26). The maturational increase in the magnitude of the flow-induced increase in [Ca2+]i in CCD cells (Fig. 3) is thus of particular interest. The modest response of the neonatal principal cell to flow cannot be explained by the absence of an apical central cilium; indeed, scanning electron microscopic analyses reveal that the principal cell cilia at birth are longer than those in the adult (10). Functional expression of Ca2+-conducting channels in the collecting duct may be low in early renal development (53). Studies in developing neurons suggest that IP3 receptors and Ca2+ channels appear to be present early in life but are not assembled to allow IP3-assisted, Ca2+-induced Ca2+ release (62).
In summary, our data suggest that the absence of flow-dependent K secretion
early in life is due to the developmental regulation of expression of maxi-K
channel -subunit message and protein. The temporal association between
appearance of flow-stimulated K secretion in the maturing CCD
(Fig. 1A) and
detection of message encoding slo (Figs.
4 and
5) and the immunodetectable
protein (Fig. 9) lends further
support to the premise that the maxi-K channel mediates flow-stimulated K
secretion. The expression of maxi-K channels in the apical membrane of
predominantly intercalated cells rather than principal cells suggests that
intercalated cells mediate, at least in part, flow-stimulated K secretion.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* Craig B. Woda and Nobuyuki Miyawaki contributed equally to this work.
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REFERENCES |
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