Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects

Craig B. Woda,1,* Nobuyuki Miyawaki,2,* Santhanam Ramalakshmi,3 Mohan Ramkumar,3 Raul Rojas,3 Beth Zavilowitz,1 Thomas R. Kleyman,3 and Lisa M. Satlin1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Both small-conductance secretory K and high-conductance Ca2+/stretch-activated maxi-K channels have been identified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1) blocked by TEA and charybdotoxin, inhibitors of intermediate- and high-conductance (maxi-K) Ca2+-activated K channels, and 2) associated with increases in net Na absorption and intracellular Ca2+ concentration ([Ca2+]i). The present study examined whether the absence of flow-stimulated K secretion early in life is due to a 1) limited flow-induced rise in net Na absorption and/or [Ca2+]i and/or 2) paucity of apical maxi-K channels. An approximately sixfold increase in tubular fluid flow rate in CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to an increase in net Na absorption and [Ca2+]i, similar in magnitude to the response observed in 6-wk-old tubules, but it failed to generate an increase in net K secretion. By 5 wk of age, there was a small, but significant, flow-stimulated rise in net K secretion that increased further by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flow stimulation of net K secretion in the adult CCD, confirming the identity of the maxi-K channel in this response. Maxi-K channel {alpha}-subunit message was consistently detected in single CCDs from animals >=4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the {alpha}-subunit revealed apical labeling of intercalated cells in cryosections from animals >=5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatal appearance of flow-dependent K secretion is determined by the transcriptional/translational regulation of expression of maxi-K channels. Furthermore, our studies suggest a novel function for intercalated cells in mediating flow-stimulated K secretion.

maxi-K channel; iberiotoxin; in vitro microperfusion; intracellular calcium concentration; mechanoregulation; development


NEWBORN HUMANS AND animals maintain a state of positive K balance, as is appropriate for growth, unlike their adult counterparts who are in net zero K balance (49). It is now well established that the neonatal kidney contributes to this K retention. Clearance studies provide abundant evidence for a low rate of urinary K excretion early in life (39, 49). In response to exogenous K loading, infants, like adults, can excrete K at a rate that exceeds its filtration (55), indicating the capacity for net tubular secretion. However, the rate of urinary K excretion in K-loaded infants and young animals is less than that observed in older animals (27, 29).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Female adult New Zealand White rabbits and litters of newborns <1 wk of age were obtained from Covance (Denver, PA) and housed in the Mount Sinai School of Medicine animal care facility. Newborns were allowed to remain with their mothers until weaning. Animals were fed standard rabbit chow and given free access to food and water. Rabbits were killed by intraperitoneal injection of pentobarbital sodium (100 mg/kg). All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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, 15–18 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 3–15% 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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of age on flow-stimulated net K secretion. CCDs isolated from 4-wk-old rabbits showed baseline net K secretion (5.4 ± 0.3 pmol · min1 · mm1, n = 5), albeit less than that measured in the 6-wk-old animals (10.5 ± 1.4 pmol · min1 · mm1, n = 5, P < 0.05) (Fig. 1A), when perfused at a flow rate of 1.3 ± 0.1 nl · min1 · mm1. However, an increase in tubular fluid flow rate to 6.0 ± 0.1 nl · min1 · mm1 failed to elicit an increase in net K secretion in CCDs isolated from 4-wk-old animals (Fig. 1A), as we previously reported (38). At 5 wk of age, an increase in tubular flow rate from 1.1 ± 0.1 to 6.3 ± 0.5 nl · min1 · mm1 resulted in a small, but significant, increase in net K secretion (from 6.4 ± 0.5 to 11.4 ± 1.2 pmol · min1 · mm1, n = 5, P < 0.05), a response that increased further in CCDs isolated from 6-wk-old animals (from 10.5 ± 1.4 to 27.0 ± 1.9 pmol · min1 · mm1 at flow rates of 1.3 ± 0.1 to 6.0 ± 0.2 nl · min1 · mm1, n = 5, P < 0.05) (Fig. 1A). Vte did not change significantly in any age group in response to an increase in tubular fluid flow rate (4 wk: –6.4 ± 0.8 to –4.4 ± 0.7 mV, P = 0.24; 5 wk: –7.4 ± 2.9 to –3.0 ± 1.6 mV, P = 0.15; 6 wk: –8.1 ± 1.4 to –6.6 ± 2.1 mV, P = 0.36).



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Fig. 1. Flow stimulation of net K secretion (A) and Na absorption (B) in the maturing cortical collecting duct (CCD). Net transport was measured at slow (~1 nl · min1 · mm1) and fast (~6 nl · min1 · mm1) flow rates in tubules isolated from 4 (n = 5)-, 5 (n = 5)-, and 6-wk-old (n = 5) rabbits. Although a 6-fold increase in flow rate stimulated net Na absorption at 4 wk to levels comparable to those observed at 6 wk of age, no flow-stimulated increase in net K secretion was detected at 1 mo of life. The flow-stimulated increase in K secretion first became apparent at 5 wk of age. *P < 0.05 compared with transport rate at 1 nl · min1 · mm1.

 

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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Fig. 2. Effect of luminal iberiotoxin (IBX) on flow-dependent K secretion (A) and Na absorption (B) in the adult CCD. Net transport (in pmol · min1 · mm1) was measured in the absence of toxin at slow and then fast flow rates before 50 nM iberiotoxin was added to the luminal perfusate. Iberiotoxin inhibited the flow-stimulated increase in net K secretion but not Na absorption; n = 3. *P < 0.05 compared with transport rate at 1 nl · min1 · mm1 in the absence of iberiotoxin.

 

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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Fig. 3. Effect of high flow on intracellular Ca2+ concentration ([Ca2+]i). CCDs isolated from 1- to 5-wk-old rabbits exhibited a significant increase in [Ca2+]i in both principal (PC) and intercalated (IC) cells following an acute increase in tubular fluid flow rate (associated with an increase in luminal diameter of 20–25%) (n = 3 CCDs at each age, except 5 wk old, where n = 4, *P < 0.05 compared with baseline [Ca2+]i).

 

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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Fig. 4. Maxi-K channel ({alpha}-subunit) mRNA expression in CCDs. RT-PCR of CCDs (top), using primer pairs designed to amplify the pore region of the {alpha}-subunit, revealed no sequence-specific signal until week 4 of life. Southern blot (middle) analysis of the PCR products, using a pore-specific probe, revealed transcripts in CCDs isolated from the 2-wk-old animal only on this blot (out of 4 in total). GAPDH-specific PCR products (bottom) were detected at each age confirming that degradation of the RNA did not occur. KDN, adult whole kidney.

 


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Fig. 5. Incidence of maxi-K transcripts in the CCD during postnatal maturation. Numbers in parentheses indicate the total number of GAPDH-positive CCD samples obtained at each week of postnatal life. At 1 wk of age, maxi-K message was not detected in any of 4 CCD sets sampled. By week 2 of life, 25% (1 of 4) of the CCD samples showed maxi-K amplification product. Maxi-K transcript was consistently detected in CCDs isolated from animals >4 wk old.

 

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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Fig. 6. Comparison of maxi-K channel ({alpha}-subunit) mRNA expression in CCDs and proximal tubules during postnatal life: representative Southern blot. RT-PCR of proximal tubules (P; all samples positive for GAPDH message) failed to generate maxi-K channel sequence-specific transcript at any age, even when probed on a Southern blot. Similar to the results shown in Fig. 4, Southern blot analysis of CCD PCR products (C) revealed transcripts in samples from 5- and 6-wk-old animals. A, whole kidney from an adult rabbit.

 

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 {alpha}-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 {alpha}-subunit dimer.



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Fig. 7. Immunoblot of rabbit kidney lysate probed with chicken antibody directed against the {alpha}-subunit of the maxi-K channel (lane 1) or antibody incubated with the immunizing peptide (lane 2). One-hundred micrograms of protein were loaded in each lane. Polypeptides with apparent molecular weights of 200 and 90 kDa were specifically recognized by the anti-maxi-K channel antibody.

 

Immunolocalization of maxi-K channels in the kidney. Cryosections of adult rabbit kidney were labeled with antibodies directed against the {alpha}-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 {alpha}-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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Fig. 8. Immunolocalization of maxi-K {alpha}-subunits in the adult rabbit kidney. Indirect immunofluorescence labeling of the {alpha}-subunit of maxi-K channels was performed in adult rabbit kidney as described under MATERIALS AND METHODS. Sections were also labeled with FITC-conjugated dolicos bifloris agglutinin (DBA), a principal cell marker. A, D, and G: DBA localization (green). B and E: maxi-K {alpha}-subunit localization (red). C and F: localization of both DBA (green) and maxi-K {alpha}-subunit (red). The maxi-K channel antibody exhibited a heterogeneous staining pattern in tubular profiles that coexpress DBA (i.e., collecting ducts). Maxi-K channel protein was detected along the apical membranes of DBA-negative cells (intercalated cells, arrows) in CCDs. Intracellular staining was observed in DBA-positive cells (arrowheads) as well as in DBA-negative cells (not shown). H and I: section of cortex labeled with antibody in the presence of excess immunizing peptide showed no maxi-K staining.

 

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 {alpha}-subunit.



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Fig. 9. Immunolocalization of maxi-K channel in the maturing rabbit kidney. Sections of kidney were labeled as described in the legend for Fig. 8. Maxi-K {alpha}-subunit (red) was not detected in collecting ducts [DBA-positive (green) tubular profiles] until week 5 of life. Sections of 5- and 6-wk-old kidneys showed both apical (arrows) and intracellular immunodetectable channels in CCDs. Apical membrane localization of maxi-K {alpha}-subunits in CCDs was observed in DBA-negative (i.e., intercalated) cells. Intracellular localization of maxi-K {alpha}-subunits was observed in both DBA-positive (arrowheads) and DBA-negative cells.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study demonstrate that flow-stimulated net K secretion is not detected in the maturing rabbit CCD until 5 wk of age (Fig. 1A) and follows the developmental appearance of baseline net K secretion (38) that is presumed to be mediated by the SK channel (i.e., ROMK). SK channel activity increases progressively after the first week of postnatal life (42), a developmental process that closely parallels increases in ROMK mRNA levels and expression of immunodetectable ROMK protein at the apical membrane of CCD principal cells (3, 64). The absence of flow-stimulated net K secretion early in life is not limited by the capacity of the CCD to respond to an increase in flow with augmented Na absorption or a rise in [Ca2+]i (Figs. 1B and 3). In fact, the transport and signaling pathways mediating these mechano-induced responses appear to be established at an early age.

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 {alpha}-subunit, a member of the slo family of K channels originally cloned from Drosophila, and a regulatory {beta}-subunit. Coexpression of both subunits in Xenopus laevis oocytes enhances the voltage, Ca2+, and charybdotoxin sensitivity of the channel compared with expression of the {alpha}-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 {alpha}-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 {alpha}-subunit common to all splice variants, our failure to detect any {alpha}-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 {alpha}-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 {alpha}-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.


    DISCLOSURES
 
This work was supported by National Institutes of Health (NIH) Grants DK-38470 (to L. M. Satlin) and DK-51391 (to T. R. Kleyman). C. B. Woda was supported by NIH Grant T32 HD-07537 (Training Grant in Developmental Biology of Membrane Transport; L. Satlin, PI) and N. B. Miyawaki by NIH Grant T32 DK-07757 (Renal Medicine Training Grant; P. Klotman, PI). Abstracts of this work were presented at the Annual Meetings of the American Society of Nephrology in 2001 (San Francisco, CA) and 2002 (Philadelphia, PA).


    ACKNOWLEDGMENTS
 
The authors thank J. Bruns for technical support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. M. Satlin, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1664, New York, NY 10029 (E-mail: lisa.satlin{at}mssm.edu).

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. Back


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