Dietary K+ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct
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《美国生理学杂志》
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
Division of Pediatric Nephrology, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York
ABSTRACT
The cortical collecting duct (CCD) is a final site for regulation of K+ homeostasis. CCD K+ secretion is determined by the electrochemical gradient and apical permeability to K+. Conducting secretory K+ (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K+ secretion, maxi-K channels appear to participate in flow-stimulated K+ secretion. Chronic dietary K+ loading enhances the CCD K+ secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693–710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K+ increases renal K+ excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107–116, 1998). The purpose of the present study was to test whether K+ adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K+ diet for 10–14 days. Real-time PCR quantitation of message encoding maxi-K - and 2–4-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); 1-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of -subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in -intercalated cells in HK kidneys. In summary, K+ adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical -subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.
potassium adaptation; intercalated cell; principal cell; in vitro microperfusion; H+-K+-ATPase
THE FINAL RENAL REGULATION of K+ excretion occurs in the mammalian late distal and connecting (CNT) tubules and collecting ducts (9, 13, 14, 17, 26, 29, 44, 65). Emerging evidence suggests that the late distal tubule and CNT, rather than the collecting duct, may be the primary physiological regulators of urinary K+ secretion (reviewed in Ref. 29). However, segments upstream from the collecting duct are exceedingly difficult to study functionally (e.g., by in vitro microperfusion) due to their short length or highly branched nature. To the extent that the cortical collecting duct (CCD) is a straight, unbranched segment and is amenable to functional analysis by in vitro microperfusion, we and others (9, 14, 44, 51, 62) have utilized this segment as a model of a K+ secretory epithelium. CCDs microperfused in vitro at physiologically slow flow rates secrete net K+ at high rates (9, 14, 26, 44). Transepithelial net K+ secretion can be further stimulated as the tubular fluid flow rate is increased (9, 14, 26, 44).
The CCD is a heterogeneous structure, composed of two morphologically and functionally distinct cell populations. The direction and magnitude of net K+ transport in this segment reflect the balance of simultaneous secretion and absorption, opposing processes considered to be mediated by principal and intercalated cell types, respectively (6, 22, 43). K+ secretion by the CCD is mediated by apical K+-selective channels. The predominant conducting apical channel in principal cell-attached patches is the inwardly rectifying, ATP-sensitive secretory K+ (SK) channel encoded by ROMK (10, 13, 58). Less easily detected in cell-attached patches of the apical membrane of the CCD is the high-conductance, Ca2+- and stretch-activated maxi-K channel (35, 45). Whereas the SK/ROMK channel is present solely in principal cells within the CCD, conducting maxi-K channels have been identified in both principal and intercalated cells in this segment (35, 45). In fact, the density of maxi-K channels in intercalated cells exceeds that detected in principal cells (35). Emerging evidence suggests that baseline K+ secretion is mediated by the SK/ROMK channel, whereas flow-stimulated K+ secretion is mediated by the maxi-K channel (2, 64).
The maxi-K channel is generally composed of both a pore-forming -subunit, a member of the slo family of K+ channels originally cloned from Drosophila melanogaster, and a regulatory -subunit (1, 8, 21). Two isoforms of the maxi-K -subunit (rbslo 1 and 2) are expressed in rabbit kidney (31, 56). Rbslo1 is expressed in the apical membrane, whereas rbslo2 is predominantly intracellular (56). Our group (64) has previously reported that the immunodetectable maxi-K channel -subunit is present along the apical membrane of intercalated cells in adult rabbits; principal cell labeling was generally intracellular and punctate in the same tubular profiles. Four maxi-K channel -subunits have been identified, and mRNA encoding each is present in mammalian kidney (39, 54, 59). The precise intrarenal distribution of the individual -subunits and, specifically, their localization within discrete tubular segments have not been explored. A role for the 1-subunit in flow-stimulated urinary K+ secretion was recently implicated by the finding that the fractional K+ excretion in maxi-K 1-null mice (BKCa-1–/–) subject to acute volume expansion was lower than that measured in wild-type animals (40). Although this observation was consistent with a low K+ secretory capacity of the CCD, these same authors more recently reported that renal 1 expression may be limited to the CNT (41), an observation that underscores the importance of the CNT in the final regulation of renal K+ secretion.
Chronic (10 days) dietary K+ loading enhances the ability of the mammalian kidney to secrete K+ (49, 65), an adaptation that includes increases in the density of SK channels and the electrochemical driving force favoring K+ exit across the apical membrane (36, 58). The observation that chronic (14 day) exposure of Ambystoma tigrinum collecting ducts to a high ambient K+ environment dramatically increases the density of apical maxi-K and epithelial Na+ (ENaC) channels (53) led us to hypothesize that K+ adaptation in response to chronic dietary manipulation is associated with an upregulation of maxi-K channel expression in the mammalian CCD. The purpose of the present study was to test this hypothesis and identify which maxi-K channel subunits are regulated by dietary K+ intake. We sought to study the effects of chronic dietary manipulations as we considered them to be physiologically relevant.
METHODS
Animals. Female New Zealand White rabbits obtained from Covance (Denver, PA) were housed in the Mount Sinai School of Medicine Center for Comparative Medicine. Rabbits were randomized to receive a control (CK; K+ content of 1%, or 256 meq/kg), low (LK; 0.13%, or 34 meq K+/kg), or high K+ (HK; 1.56%, or 398 meq/kg) diet, all containing 0.29% Na+ or 7.5 gm/kg diet (modification of TD87433; Harlan Teklad, Madison, WI) for 10–14 days. All rabbits were allowed free access to water and their modified diets. Animals were euthanized in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. At the time of death, samples of ventricular blood (serum) and urine were obtained for measurement of electrolyte concentration. Animal protocols were approved by the Institutional Animal Care and Use Committee.
Microperfusion of single tubules for measurement of net cation transport rates. CCDs microdissected from LK and HK rabbit kidneys were microperfused in vitro, and tubular fluid samples were collected at both slow physiological (1 nl·min–1·mm–1) and fast (5 nl·min–1·mm–1) flow rates. Tubules were 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 sodium lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (62, 64). 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). Collected samples were analyzed for K+ and Na+ concentrations by helium glow photometry, and the rates of net K+ secretion and Na+ absorption were calculated as previously described (44, 64).
Maxi-K channel primers and probes. Gene-specific primers and probes for maxi-K - and 1–4-subunits were designed using Primer Express software (Applied Biosystems), following the recommended guidelines based on sequences obtained from GenBank. The sequences of primers and probes are listed in Table 1. The primers for the - and 3-subunits were designed to amplify highly conserved regions of each, common to all splice variants. TaqMan probes (for , 1, 2, 4) were labeled with the fluorescent reporter dye 6-carboxyfluorescein at the 5' end and the quencher dye 6-carboxy-tetramethylrhodamine at the 3' end. The specificity of primer pairs was confirmed by agarose gel electrophoresis and sequencing of PCR products.
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Relative quantitation of maxi-K channel subunit genes in CCD. For molecular analysis of single rabbit CCDs, tubule microdissection was carried out in 1x PBS containing 10 mM vanadyl ribonucleoside complexes (Sigma, St. Louis, MO) for no longer than 1 h after the death of the animal. Approximately 10-mm total lengths of CCDs were pooled for each sample. RNA was extracted from the single CCDs and cDNA synthesized using oligo(dT) primers (as described in Ref. 64).
Channel transcript expression was analyzed by real-time semi-quantitative PCR using TaqMan (for detection of -, 1-, 2-, and 4-subunits) or SYBR green [for detection of 3-subunit and colonic and gastric H+-K+-ATPases (HKAc and HKAg, respectively)] techniques and an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). In this system, accumulation of PCR products was detected by monitoring the increase in fluorescence of the reporter dye from the TaqMan probes or double-strand DNA binding SYBR green.
For the TaqMan assays, to each well of a 384-well plate was added 2 μl of cDNA and 8 μl of PCR Master Mix, including 0.05 μl Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), AmpErase UNG (Applied Biosystems), dNTPs with dUTP (Applied Biosystems), passive reference ROX (Invitrogen), optimized buffer component (0.2 μl; 20 pM), forward and reverse primers, 0.2 μl of TaqMan probe, and nuclease-free water (Promega) (total volume 10 μl). Each plate was then capped; after the initial steps of 50°C/2 min for optimal AmpErase UNG enzyme activity and 95°C/10 min for activation of DNA polymerase, 40 cycles of 95°C/15 s (melt) and 60°C/1 min (anneal/extend) were performed. The resulting data were analyzed by using SDS 2.1 software (Applied Biosystems). For SYBR green assays (3-subunit as well as HKA), each well contained 2 μl of cDNA, 30 pmol forward and reverse primers, 8 μl of SYBR green PCR Master Mix (Qiagen, Valencia, CA), and water to a total volume of 20 μl; wells were sealed with an optical adhesive cover and run at 95°C/5 min and then at 94°C/15 s, 55°C/30 s, and finally 72°C/30 s for 40 cycles.
PCR of CCD samples that were isolated from CK, LK, and HK rabbits on the same day were run in quadruplicate on the same plate to minimize interassay variation. For each CCD sample, the relative amounts of each of five target genes and 18S, the latter run on a separate plate with 18S rRNA standards and primers, were estimated using the standard curve method (60). An 18S rRNA control kit (Applied Biosystems) was used for internal quantification. The ABI 7900 detection system records the number of PCR cycles (Ct) required for fluorescence (i.e., product) to reach a threshold value. The standard curve for 18S plots Ct values vs. masses of RNA analyzed. The standard curve for the target gene plots Ct values vs. an arbitrary "fold" expression (Fig. 1). Relative gene expression was calculated for each sample by first solving for RNA mass, represented by 18S expression using the standard curve. The expression of the target gene was then normalized to RNA mass determined from 18S expression.
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 subjected to 4–15% 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 sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 10 mM KCl, pH 7.4) plus 0.05% Tween 20 and then probed with the affinity-purified anti--subunit antibody, raised in chicken against the sequence NQYKSTSSLIPPIREVEDEC corresponding to a COOH-terminal region of mouse maxi-K -subunit (Aves Labs) (64), at a concentration of 0.5 μg/ml in PBS-Tween 20 with 5% milk 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 after incubation with a 1:2,500 dilution of horseradish peroxidase-conjugated goat anti-chicken IgG (Kierkegaard and Perry Laboratories, Gaithersburg, MD) by chemiluminescence (Western blot chemiluminescence reagent plus; New England Nuclear, Boston, MA).
Immunofluorescence localization of maxi-K channels in kidney. Coronal sections of rabbit kidneys were fixed in 4% paraformaldehyde and sucrose and embedded in paraffin. Serial 4-μm-thick paraffin 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 a PBS solution (PBS, 0.02% glycine, and 5% milk). The tissue was then permeabilized with PBS solution with 0.1% Triton X-100 for 10 min and then blocked with PBS solution for 30 min.
Tissue sections were incubated with the anti-maxi-K channel -subunit antibody (15 μg/ml) for 1 h at room temperature. For peptide competition experiments, peptide was added to the primary antibody at a 1:100 dilution (stock concentration of 10.5 mg/ml). After incubation with primary antibody, the sections were washed three times for 5 min with PBS solution. The secondary anti-chicken IgY antibody, a Cy5-conjugated Affinipure F(ab')2 fragment from donkey, was applied at a 1:50 dilution (stock concentration of 1.5 mg/ml) and sections were colabeled with the rabbit principal cell marker (64) fluorescein Dolichos biflorus agglutinin (DBA; 5 μg/ml) in PBS solution for 45 min at room temperature. Alternatively, sections were colabeled with the -intercalated cell marker fluorescein peanut agglutinin (5 μg/ml in PBS) (63). Each section was washed three times with PBS solution for 5 min followed by three rapid washes with PBS. H+ pump was immunolocalized using an anti-E11 antibody (gift from Stephen Gluck) in a 1:1 dilution. The secondary antibody, a Cy3-conjugated Affinipure F(ab')2 fragment goat anti-mouse IgG, was applied at 1:100 dilution.
All sections were mounted on coverslips with phenylene diamine mounting media. Confocal microscopy was performed on a Leica DMRXE equipped with krypton, argon, and helium-neon lasers. Images were acquired with the use of a x100 plan-apochromat objective (numerical aperture 1.4) and appropriate filter combination. Settings were as follows: photomultipliers set to 600–800 mV, 1.0-μm pinhole, zoom of 2.25, and Kalman filter (n = 4). The images (512 x 512 pixels) were saved in a tag-information-file-format (TIFF), and the contrast levels of the images were adjusted in the Photoshop program (Adobe, Mountain View, CA) on a 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, with n = the number of animal or tubule samples used for in vitro microperfusion studies and PCR. Comparisons were made by paired and unpaired t-tests as appropriate, using commercially available statistical software for the calculations (SPSS, Chicago, IL). Data comparisons among the three dietary groups (chemistries, real-time PCR) were performed by ANOVA with Tukey's post hoc test. Significance was asserted at P < 0.05.
RESULTS
Effect of diet on serum and urine electrolytes. Table 2 lists the serum and urine Na+ and K+ concentrations measured in 4–10 animals on each of the diets. There were no significant differences in serum or urine Na+ concentrations among the three groups. Predictably, the serum and urine K+ concentrations differed between animals on the three diets. The differences in weight gain among the three dietary groups were not statistically significant by ANOVA.
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Effects of diet on flow-stimulated net K+ secretion. At a slow flow rate of 1 nl·min–1·mm–1, the rate of net K+ secretion (in pmol·min–1·mm–1) in CCDs isolated from LK animals did not differ from that measured in HK animals (Fig. 2A). An increase in tubular fluid flow rate to 5 nl·min–1·mm–1 elicited an almost threefold stimulation in the rate of net K+ secretion in CCDs isolated from HK animals. CCDs from LK animals failed to exhibit a flow response (P = not significant) (Fig. 2A).
Net Na+ absorption was measured in the same tubules as described directly above. As shown in Fig. 2B, at a flow rate of 1 nl·min–1·mm–1, net Na+ absorption was lower in LK compared with HK animals (P < 0.01), presumably reflecting a state of hypoaldosteronism associated with K+ depletion. After an increase in tubular flow rate, net Na+ absorption increased significantly in both HK and LK animals. Although an increase in net Na+ absorption would be expected to increase the driving force for K+ secretion, this was not observed. We have previously reported that an increase in luminal flow rate in the CCD leads to an increase in net Na+ absorption that is unaccompanied by an increase in the lumen negative transepithelial voltage (44, 46, 62, 64). We have speculated in the past (46) that an increase in luminal flow rate enhances the paracellular permeability to Cl–, leading to movement of negative charge out of the lumen.
In two experiments using CCDs obtained from HK animals, luminal addition of 50 nM iberiotoxin inhibited flow-stimulated net K+ secretion (from 18.7 to 4.9 pmol·min–1·mm–1 and 15.6 to 4.1 pmol·min–1·mm–1) but was without consistent effect on net Na+ absorption (from 92.2 to 116.2 pmol·min–1·mm–1 and from 80.0 to 57.8 pmol·min–1·mm–1). These results, identical to those reported by us previously (64), are consistent with the notion that flow-stimulated K+ secretion is mediated by an iberiotoxin-sensitive maxi-K channel.
Effect of diet on expression of maxi-K channel subunit mRNA. The relative abundance of maxi-K channel - and 1–4-subunit transcripts was examined by real-time PCR analysis of whole kidney from LK and HK rabbits (Fig. 3) and single CCDs isolated from CK, LK, and HK rabbits (Fig. 4). All of the maxi-K channel subunits examined were expressed in whole kidney, and there were no significant differences in message levels in kidneys isolated from LK and HK animals (Fig. 3). Single CCDs expressed maxi-K - and 2–4-subunit mRNAs. However, 1 message was not detected in any -actin-positive CCD (Fig. 4), although this transcript was expressed in whole kidney (Fig. 3). Maxi-K - and 2–4 mRNA expression in CCDs isolated from HK animals was significantly greater than that detected in CK animals (P < 0.05) (Fig. 4). Conversely, maxi-K - and 2–4-subunit mRNA expression in CCDs isolated from LK animals was significantly less than that detected in CK animals (P < 0.05) (Fig. 4)
To confirm that the HK diet-induced increase in expression of maxi-K transcripts tested was specific to this channel and not due to a general stimulatory effect of K+ on transcription, the effect of dietary K+ intake on HKAc and HKAg -subunit transcript abundance was also determined. HKAc was markedly stimulated in CCDs from LK animals, whereas HKAg was significantly reduced in this experimental group (P < 0.05) (Fig. 5). The HK diet had no effect on expression of either HKA compared with the levels of expression detected in CK animals.
Western analysis of maxi-K channel protein in kidney homogenate. An immunoblot of whole rabbit kidney lysates prepared from CK, LK, and HK rabbits (n = 3 kidneys for each dietary group) was performed to discern whether dietary K+ intake led to changes in protein abundance at the level of the whole kidney (Fig. 6). Polypeptides with apparent molecular masses of 90 kDa were recognized by the antibody but not by antibody preincubated with the immunizing peptide (64). There was no apparent difference in abundance of whole kidney maxi-K expression among the three groups.
Effect of diet on immunolocalization of maxi-K channel -subunit. Given our concern that 1) diet-induced alterations in maxi-K channel expression in a subset of nephron segments, which represents only a minor fraction of the total renal mass, might be missed by Western blotting of whole kidney and 2) these studies do not address the cellular localization of channels, we sought to determine whether maxi-K channel -subunit expression and localization in single CCDs are regulated by diet. To examine this possibility, cryosections of rabbit kidneys were labeled with the antibody directed against the -subunit of the maxi-K channel and the principal cell-specific apical marker DBA and examined by indirect immunofluorescence microscopy.
In animals fed an HK diet, immunodetectable -subunit was detected in a linear pattern along the apical membrane (Figs. 7–9). Apical membrane maxi-K -subunit expression was limited primarily to -intercalated cells (Figs. 7–9), as the channel was present in cells expressing immunodetectable H+-ATPase (Fig. 8) and was only occasionally observed in cells that were labeled with peanut agglutinin, a -intercalated cell marker (Fig. 9). Apical labeling for -subunit was rarely observed in CCDs from animals fed a LK diet (Fig. 7). In the latter and CK animals, the distribution of immunodetectable channel protein appeared to be more diffuse and detected in a predominantly intracellular distribution, compared with CCDs from animals maintained on the HK diet.
The numbers of DBA-positive and DBA-negative cells, defined as principal and intercalated cells, respectively, and the number of DBA-negative (intercalated) cells showing linear apical maxi-K -subunit localization were counted in individual tubular profiles. Linear apical channel localization, not observed in DBA-positive cells, was present in 9% of intercalated cells in CK kidney sections and 29% of intercalated cells in HK animals (Table 3) (n 3 kidneys for each dietary group).
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DISCUSSION
The results of the present study indicate that dietary K+ intake regulates the capacity of the rabbit CCD for flow-stimulated K+ secretion, a transport process our group (62, 64) and others (2, 25) have proposed to be mediated by the maxi-K channel. It is well established that net K+ secretion and the density of apical conducting SK/ROMK channels in the CCD are exquisitely regulated in response to physiological demands, as reviewed in this discussion. The present study is, to our knowledge, the first to identify a contribution of the maxi-K channel to renal K+ adaptation in the mammalian CCD, a response that includes changes in steady-state abundance of maxi-K channel - and 2–4-subunit mRNAs (Fig. 4) as well as cellular localization of channel proteins in the CCD (Fig. 7). The increase in surface expression of maxi-K channels that we observed in response to an increase in dietary K+ intake is consistent with an increase in whole cell expression of maxi-K channels and/or a redistribution of channels from an intracellular pool to the plasma membrane.
Wang et al. (56) reported that COOH-terminal splice variants of maxi-K -subunit exhibit differences in cellular localization. Although our study does not address potential mechanisms that lead to a redistribution of K+ channels from an intracellular pool to the plasma membrane in response to an increase in dietary K+, it is possible that alternative splicing of the COOH terminus of maxi-K -subunit may contribute to the differences in cellular localization of maxi-K channel that we observed in animals maintained on different K+ diets (Fig. 7). The anti-maxi-K channel antibody that we used is directed against a conserved COOH-terminal domain of the -subunit that is present in -subunits cloned from rabbit kidney (GenBank no. BAA23747), as well as -subunits isolated from mouse, human, and dog. We are aware that this epitope resides in a region where alternative splicing may result in loss of this sequence, and thus not all splice variants may be detected with this antibody.
Cumulative evidence suggests that K+ adaptation in the rat is due, at least in part, to an increase in density of conducting SK channels in the apical membrane of the CCD (36, 37, 57, 58). The observation that the number of SK channels is not altered in CCDs isolated from rats on a low-Na+ diet, a maneuver that increases the circulating levels of aldosterone, suggests that the effect of HK intake on SK number is not mediated by this hormone (36). Furthermore, Frindt et al. (11) reported that an HK diet does not increase transcription of SK channels, since the relative amounts of ROMK mRNA were the same in single CCDs isolated from animals on HK and CK diets. The latter investigators thus proposed that the effect of an HK diet on the number of SK channels in rat CCD is achieved by a posttranslational modulation of a preexisting pool of channels or closely associated proteins (11). It should be noted that, in rabbits subjected to K+ loading, baseline net K+ secretion measured at the slow flow rate of 1 nl·min–1·mm–1, presumably mediated by the SK channel, was not statistically different from that observed in tubules isolated from LK animals (Fig. 2A). Whether this reflects a limited capacity of the rabbit vs. the rat to augment SK channel expression at the apical membrane of the CCD remains to be determined.
Controversy exists as to whether chronic K+ restriction decreases the number of conducting SK channels in the apical membrane of the rat CCD (37, 57). However, 2–5 days of LK intake has been shown to decrease the abundance of ROMK in both cortex and medulla (30) and specifically in the CCD, presumably due to an increase in endocytosis and degradation of the channel (5). Conservation of K+ and K+ reabsorption by the CCD may be further facilitated in the face of K+ restriction by upregulation of renal HKA, specifically at the apical membrane of the collecting duct (48). Both HKAg and HKAc transporters are expressed in the kidney but are differentially regulated (reviewed in Ref. 48). HKAc mRNA is upregulated in cortex of K+-depleted rats (32), whereas HKAg mRNA and protein abundance remain essentially unchanged in kidney under these same conditions (23). In the present study, we confirm, at the level of the single CCD, that K+ depletion induces mRNA encoding the -subunit of HKAc, which presumably facilitates K+ absorption from the tubular fluid (Fig. 5).
The results of the present study reveal that a fivefold increase in flow rate stimulated net K+ secretion only in CCDs isolated from HK but not LK animals (Fig. 2). The adaptation of the rabbit CCD to the HK diet is consistent with an increase in the apical K+ conductance and, based on studies in rats, likely reflects, at least in part, an increase in the number of active SK channels on the apical membrane. However, the dietary K+-induced increase in expression of mRNA encoding maxi-K - and 2–4-subunits, apical immunodetectable maxi-K -subunit in the CCD, and the sensitivity of the flow-induced increase in net K+ secretion to iberiotoxin provide compelling evidence for a major contribution of the maxi-K channel to the K+-adaptive response of the rabbit CCD.
Plasma aldosterone levels were not measured in the present study. However, on the basis of measurements from studies by others (61) in which rabbits were fed HK and LK diets within ranges of those utilized in the present study, it seems reasonable to assume that plasma aldosterone concentrations were greater in animals ingesting the HK compared with LK diets. In fact, we consider that low circulating levels of aldosterone in the LK rabbits accounts for the blunted rates of net Na+ absorption in CCDs isolated from this experimental group (Fig. 2). Low plasma concentrations of aldosterone are associated with undetectable apical ENaC activity in principal cells, as measured by patch-clamp analysis (34), and absence of immunodetectable ENaC subunits on the apical membrane of cells lining the aldosterone-sensitive distal nephron (24).
Conversely, the stimulation of net K+ secretion in HK animals (Fig. 2) may also be explained by an aldosterone-induced increase in the apical expression of conducting Na+ channels (24, 34) and net Na+ absorption (47), leading to an enhanced electrochemical diving force for K+ secretion into the urinary fluid. Aldosterone may also activate other intracellular second messenger systems that regulate maxi-K channel expression and activity. Aldosterone-induced stimulation of electrogenic Na+ transport in A6 renal cells may be dependent on a Ca2+/calmodulin-dependent protein kinase, as suggested by the sensitivity of the aldosterone-induced short-circuit current to W-7 and trifluoperazine (38). Ca2+/calmodulin-dependent protein kinase IV signaling has been implicated as pivotal in the regulation of alternative splicing of maxi-K pre-mRNAs through a defined RNA regulatory element, the Ca2+/calmodulin-dependent protein kinase IV-responsive RNA element (67). These observations suggest that Ca2+/calmodulin-dependent protein kinase may link increases in intracellular Ca2+ concentrations to transcriptional regulation of genes. Finally, CCD maxi-K channel activity is highly pH sensitive at physiological low cytosolic Ca2+ activities (16). To the extent that hyperaldosteronism is associated with metabolic alkalosis (12), the acid-base disturbance may have a direct causal role in increasing apical K+ channel activity during dietary K+ loading.
Patch-clamp analysis indicates that K+ loading stimulates maxi-K channel activity in rat distal colon (4). The effects of dietary K+ loading on maxi-K channel expression have not been rigorously examined in the mammalian CCD. However, long-term exposure (>7 days) of the amphibian Ambystoma tigrinum collecting duct to elevated environmental K+ increases their rate of renal K+ excretion (50). This adaptation is due, in part, to increases in apical maxi-K (10-fold) and ENaC (4-fold) channel density in collecting ducts isolated from these animals (53). Whereas these segments do not routinely exhibit maxi-K channel activity when studied at their resting membrane potential in 4 mM peritubular K+, collecting ducts bathed with a 15 mM K+ solution exhibit a high density of conducting maxi-K channels (52).
The present study is the first to characterize the identity of maxi-K -subunit expression in the mammalian CCD. The presence of 1 message in CNT (41) and its absence in all CCD samples studied in the present investigation suggest that -subunit expression is differentially regulated along the nephron. We now show that mRNA encoding 2–4 is present in the CCD and that the steady-state abundance of each subunit transcript is increased in response to dietary K+ loading when compared with levels in CCDs from animals on a CK diet. We acknowledge that our data do not address whether 2–4 protein is translated and expressed with the -subunit as a functional channel. Although single cells may contain multiple -subunit variants (19, 33, 42), our analysis of native tubules of heterogeneous cellular composition does not allow us to draw any conclusions about whether multiple -subunits are expressed in single cells.
The tissue-specific expression of -subunits has been proposed to allow for the assembly of a large number of distinct maxi-K channels in vivo, contributing to the functional diversity of native BK currents. In heterologous systems, coexpression of - with -subunits alters the voltage, Ca2+, and inhibitor sensitivity of the channel compared with expression of the -subunit alone. Specifically, coexpression of 1 with increases Ca2+ sensitivity, slows gating kinetics, and alters pharmacological properties of the channel (7, 20, 27). Coexpression of 2 with results in rapid and complete inactivation of channel currents (54, 55). The 3 partially inactivates maxi-K current and induces inward rectification of the current-voltage relationship (3, 54, 66); 4 increases the sensitivity for voltage, alters the gating behavior of the expressed channels in a Ca2+-dependent manner, and, if glycosylated, dramatically reduces iberiotoxin association rates (3, 15, 18, 28). On the basis of the inhibitory nature of the 2- and 3-subunits, it is unlikely that these subunits contribute significantly to the maxi-K conductance in native CCD. Whether the native channel is comprised of an -subunit alone, an -subunit associated with a nonglycosylated (iberiotoxin-sensitive) 4-subunit (18), or an as yet undescribed -subunit has yet to be determined.
The results of the present investigation do not allow us to discern whether the increases in dietary K+ load, plasma and/or cellular K+ concentration, and/or levels of circulating aldosterone stimulate de novo synthesis of new K+ channel protein(s) or the recruitment of preexisting K+ channels from within the cytosol to the apical membrane.
To begin to address this question, future efforts will need to investigate whether dietary Na+ depletion, which induces an even greater degree of secondary hyperaldosteronism than does K+ loading, elicits similar increases in maxi-K channel message, protein localization, and net K+ secretion compared with results reported in the present study.
In summary, our results provide compelling evidence to support the hypothesis that K+ adaptation to dietary K+ loading is associated with an increase in apical maxi-K channel activity in the mammalian CCD. Specifically, we now report that an increase in dietary K+ intake leads to increases in maxi-K channel - and 2–4-subunit mRNA expression and immunodetectable apical -subunit in the CCD. These results suggest that the adaptive response of the CCD to K+ loading includes transcriptional regulation of message and de novo synthesis of new maxi-K channel subunits, as well as the redistribution of channels from an intracellular pool to the plasma membrane, in sum leading to an increase in functional channel expression. We speculate that these adaptive responses, in concert with those already well described for the SK/ROMK channel, contribute to the maintenance of K+ homeostasis under conditions of K+ loading.
GRANTS
This work was supported by National Institutes of Health (NIH) Grants DK-038470 (to L. M. Satlin) and DK-051391 (to T. R. Kleyman). H. Zhou was supported by NIH Grant T32 HD-07537 (Training Grant in Developmental Biology of Membrane Transport; L. Satlin, principal investigator). T. Morimoto was supported by a Kidney and Urology Foundation of America Fellowship Grant.
Abstracts of this work were presented at the Annual Meetings of the American Society of Nephrology in 2003 (San Diego, CA) and 2004 (St. Louis, MO).
ACKNOWLEDGMENTS
The authors gratefully acknowledge Beth Zavilowitz for technical support and the MSSM Quantitative PCR Shared Research Facility.
FOOTNOTES
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.
F. Najjar and H. Zhou contributed equally to this work.
T. R. Kleyman and L. M. Satlin contributed equally to this work.
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Division of Pediatric Nephrology, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York
ABSTRACT
The cortical collecting duct (CCD) is a final site for regulation of K+ homeostasis. CCD K+ secretion is determined by the electrochemical gradient and apical permeability to K+. Conducting secretory K+ (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K+ secretion, maxi-K channels appear to participate in flow-stimulated K+ secretion. Chronic dietary K+ loading enhances the CCD K+ secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693–710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K+ increases renal K+ excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107–116, 1998). The purpose of the present study was to test whether K+ adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K+ diet for 10–14 days. Real-time PCR quantitation of message encoding maxi-K - and 2–4-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); 1-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of -subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in -intercalated cells in HK kidneys. In summary, K+ adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical -subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.
potassium adaptation; intercalated cell; principal cell; in vitro microperfusion; H+-K+-ATPase
THE FINAL RENAL REGULATION of K+ excretion occurs in the mammalian late distal and connecting (CNT) tubules and collecting ducts (9, 13, 14, 17, 26, 29, 44, 65). Emerging evidence suggests that the late distal tubule and CNT, rather than the collecting duct, may be the primary physiological regulators of urinary K+ secretion (reviewed in Ref. 29). However, segments upstream from the collecting duct are exceedingly difficult to study functionally (e.g., by in vitro microperfusion) due to their short length or highly branched nature. To the extent that the cortical collecting duct (CCD) is a straight, unbranched segment and is amenable to functional analysis by in vitro microperfusion, we and others (9, 14, 44, 51, 62) have utilized this segment as a model of a K+ secretory epithelium. CCDs microperfused in vitro at physiologically slow flow rates secrete net K+ at high rates (9, 14, 26, 44). Transepithelial net K+ secretion can be further stimulated as the tubular fluid flow rate is increased (9, 14, 26, 44).
The CCD is a heterogeneous structure, composed of two morphologically and functionally distinct cell populations. The direction and magnitude of net K+ transport in this segment reflect the balance of simultaneous secretion and absorption, opposing processes considered to be mediated by principal and intercalated cell types, respectively (6, 22, 43). K+ secretion by the CCD is mediated by apical K+-selective channels. The predominant conducting apical channel in principal cell-attached patches is the inwardly rectifying, ATP-sensitive secretory K+ (SK) channel encoded by ROMK (10, 13, 58). Less easily detected in cell-attached patches of the apical membrane of the CCD is the high-conductance, Ca2+- and stretch-activated maxi-K channel (35, 45). Whereas the SK/ROMK channel is present solely in principal cells within the CCD, conducting maxi-K channels have been identified in both principal and intercalated cells in this segment (35, 45). In fact, the density of maxi-K channels in intercalated cells exceeds that detected in principal cells (35). Emerging evidence suggests that baseline K+ secretion is mediated by the SK/ROMK channel, whereas flow-stimulated K+ secretion is mediated by the maxi-K channel (2, 64).
The maxi-K channel is generally composed of both a pore-forming -subunit, a member of the slo family of K+ channels originally cloned from Drosophila melanogaster, and a regulatory -subunit (1, 8, 21). Two isoforms of the maxi-K -subunit (rbslo 1 and 2) are expressed in rabbit kidney (31, 56). Rbslo1 is expressed in the apical membrane, whereas rbslo2 is predominantly intracellular (56). Our group (64) has previously reported that the immunodetectable maxi-K channel -subunit is present along the apical membrane of intercalated cells in adult rabbits; principal cell labeling was generally intracellular and punctate in the same tubular profiles. Four maxi-K channel -subunits have been identified, and mRNA encoding each is present in mammalian kidney (39, 54, 59). The precise intrarenal distribution of the individual -subunits and, specifically, their localization within discrete tubular segments have not been explored. A role for the 1-subunit in flow-stimulated urinary K+ secretion was recently implicated by the finding that the fractional K+ excretion in maxi-K 1-null mice (BKCa-1–/–) subject to acute volume expansion was lower than that measured in wild-type animals (40). Although this observation was consistent with a low K+ secretory capacity of the CCD, these same authors more recently reported that renal 1 expression may be limited to the CNT (41), an observation that underscores the importance of the CNT in the final regulation of renal K+ secretion.
Chronic (10 days) dietary K+ loading enhances the ability of the mammalian kidney to secrete K+ (49, 65), an adaptation that includes increases in the density of SK channels and the electrochemical driving force favoring K+ exit across the apical membrane (36, 58). The observation that chronic (14 day) exposure of Ambystoma tigrinum collecting ducts to a high ambient K+ environment dramatically increases the density of apical maxi-K and epithelial Na+ (ENaC) channels (53) led us to hypothesize that K+ adaptation in response to chronic dietary manipulation is associated with an upregulation of maxi-K channel expression in the mammalian CCD. The purpose of the present study was to test this hypothesis and identify which maxi-K channel subunits are regulated by dietary K+ intake. We sought to study the effects of chronic dietary manipulations as we considered them to be physiologically relevant.
METHODS
Animals. Female New Zealand White rabbits obtained from Covance (Denver, PA) were housed in the Mount Sinai School of Medicine Center for Comparative Medicine. Rabbits were randomized to receive a control (CK; K+ content of 1%, or 256 meq/kg), low (LK; 0.13%, or 34 meq K+/kg), or high K+ (HK; 1.56%, or 398 meq/kg) diet, all containing 0.29% Na+ or 7.5 gm/kg diet (modification of TD87433; Harlan Teklad, Madison, WI) for 10–14 days. All rabbits were allowed free access to water and their modified diets. Animals were euthanized in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. At the time of death, samples of ventricular blood (serum) and urine were obtained for measurement of electrolyte concentration. Animal protocols were approved by the Institutional Animal Care and Use Committee.
Microperfusion of single tubules for measurement of net cation transport rates. CCDs microdissected from LK and HK rabbit kidneys were microperfused in vitro, and tubular fluid samples were collected at both slow physiological (1 nl·min–1·mm–1) and fast (5 nl·min–1·mm–1) flow rates. Tubules were 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 sodium lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (62, 64). 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). Collected samples were analyzed for K+ and Na+ concentrations by helium glow photometry, and the rates of net K+ secretion and Na+ absorption were calculated as previously described (44, 64).
Maxi-K channel primers and probes. Gene-specific primers and probes for maxi-K - and 1–4-subunits were designed using Primer Express software (Applied Biosystems), following the recommended guidelines based on sequences obtained from GenBank. The sequences of primers and probes are listed in Table 1. The primers for the - and 3-subunits were designed to amplify highly conserved regions of each, common to all splice variants. TaqMan probes (for , 1, 2, 4) were labeled with the fluorescent reporter dye 6-carboxyfluorescein at the 5' end and the quencher dye 6-carboxy-tetramethylrhodamine at the 3' end. The specificity of primer pairs was confirmed by agarose gel electrophoresis and sequencing of PCR products.
View this table:
Relative quantitation of maxi-K channel subunit genes in CCD. For molecular analysis of single rabbit CCDs, tubule microdissection was carried out in 1x PBS containing 10 mM vanadyl ribonucleoside complexes (Sigma, St. Louis, MO) for no longer than 1 h after the death of the animal. Approximately 10-mm total lengths of CCDs were pooled for each sample. RNA was extracted from the single CCDs and cDNA synthesized using oligo(dT) primers (as described in Ref. 64).
Channel transcript expression was analyzed by real-time semi-quantitative PCR using TaqMan (for detection of -, 1-, 2-, and 4-subunits) or SYBR green [for detection of 3-subunit and colonic and gastric H+-K+-ATPases (HKAc and HKAg, respectively)] techniques and an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). In this system, accumulation of PCR products was detected by monitoring the increase in fluorescence of the reporter dye from the TaqMan probes or double-strand DNA binding SYBR green.
For the TaqMan assays, to each well of a 384-well plate was added 2 μl of cDNA and 8 μl of PCR Master Mix, including 0.05 μl Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), AmpErase UNG (Applied Biosystems), dNTPs with dUTP (Applied Biosystems), passive reference ROX (Invitrogen), optimized buffer component (0.2 μl; 20 pM), forward and reverse primers, 0.2 μl of TaqMan probe, and nuclease-free water (Promega) (total volume 10 μl). Each plate was then capped; after the initial steps of 50°C/2 min for optimal AmpErase UNG enzyme activity and 95°C/10 min for activation of DNA polymerase, 40 cycles of 95°C/15 s (melt) and 60°C/1 min (anneal/extend) were performed. The resulting data were analyzed by using SDS 2.1 software (Applied Biosystems). For SYBR green assays (3-subunit as well as HKA), each well contained 2 μl of cDNA, 30 pmol forward and reverse primers, 8 μl of SYBR green PCR Master Mix (Qiagen, Valencia, CA), and water to a total volume of 20 μl; wells were sealed with an optical adhesive cover and run at 95°C/5 min and then at 94°C/15 s, 55°C/30 s, and finally 72°C/30 s for 40 cycles.
PCR of CCD samples that were isolated from CK, LK, and HK rabbits on the same day were run in quadruplicate on the same plate to minimize interassay variation. For each CCD sample, the relative amounts of each of five target genes and 18S, the latter run on a separate plate with 18S rRNA standards and primers, were estimated using the standard curve method (60). An 18S rRNA control kit (Applied Biosystems) was used for internal quantification. The ABI 7900 detection system records the number of PCR cycles (Ct) required for fluorescence (i.e., product) to reach a threshold value. The standard curve for 18S plots Ct values vs. masses of RNA analyzed. The standard curve for the target gene plots Ct values vs. an arbitrary "fold" expression (Fig. 1). Relative gene expression was calculated for each sample by first solving for RNA mass, represented by 18S expression using the standard curve. The expression of the target gene was then normalized to RNA mass determined from 18S expression.
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 subjected to 4–15% 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 sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 10 mM KCl, pH 7.4) plus 0.05% Tween 20 and then probed with the affinity-purified anti--subunit antibody, raised in chicken against the sequence NQYKSTSSLIPPIREVEDEC corresponding to a COOH-terminal region of mouse maxi-K -subunit (Aves Labs) (64), at a concentration of 0.5 μg/ml in PBS-Tween 20 with 5% milk 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 after incubation with a 1:2,500 dilution of horseradish peroxidase-conjugated goat anti-chicken IgG (Kierkegaard and Perry Laboratories, Gaithersburg, MD) by chemiluminescence (Western blot chemiluminescence reagent plus; New England Nuclear, Boston, MA).
Immunofluorescence localization of maxi-K channels in kidney. Coronal sections of rabbit kidneys were fixed in 4% paraformaldehyde and sucrose and embedded in paraffin. Serial 4-μm-thick paraffin 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 a PBS solution (PBS, 0.02% glycine, and 5% milk). The tissue was then permeabilized with PBS solution with 0.1% Triton X-100 for 10 min and then blocked with PBS solution for 30 min.
Tissue sections were incubated with the anti-maxi-K channel -subunit antibody (15 μg/ml) for 1 h at room temperature. For peptide competition experiments, peptide was added to the primary antibody at a 1:100 dilution (stock concentration of 10.5 mg/ml). After incubation with primary antibody, the sections were washed three times for 5 min with PBS solution. The secondary anti-chicken IgY antibody, a Cy5-conjugated Affinipure F(ab')2 fragment from donkey, was applied at a 1:50 dilution (stock concentration of 1.5 mg/ml) and sections were colabeled with the rabbit principal cell marker (64) fluorescein Dolichos biflorus agglutinin (DBA; 5 μg/ml) in PBS solution for 45 min at room temperature. Alternatively, sections were colabeled with the -intercalated cell marker fluorescein peanut agglutinin (5 μg/ml in PBS) (63). Each section was washed three times with PBS solution for 5 min followed by three rapid washes with PBS. H+ pump was immunolocalized using an anti-E11 antibody (gift from Stephen Gluck) in a 1:1 dilution. The secondary antibody, a Cy3-conjugated Affinipure F(ab')2 fragment goat anti-mouse IgG, was applied at 1:100 dilution.
All sections were mounted on coverslips with phenylene diamine mounting media. Confocal microscopy was performed on a Leica DMRXE equipped with krypton, argon, and helium-neon lasers. Images were acquired with the use of a x100 plan-apochromat objective (numerical aperture 1.4) and appropriate filter combination. Settings were as follows: photomultipliers set to 600–800 mV, 1.0-μm pinhole, zoom of 2.25, and Kalman filter (n = 4). The images (512 x 512 pixels) were saved in a tag-information-file-format (TIFF), and the contrast levels of the images were adjusted in the Photoshop program (Adobe, Mountain View, CA) on a 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, with n = the number of animal or tubule samples used for in vitro microperfusion studies and PCR. Comparisons were made by paired and unpaired t-tests as appropriate, using commercially available statistical software for the calculations (SPSS, Chicago, IL). Data comparisons among the three dietary groups (chemistries, real-time PCR) were performed by ANOVA with Tukey's post hoc test. Significance was asserted at P < 0.05.
RESULTS
Effect of diet on serum and urine electrolytes. Table 2 lists the serum and urine Na+ and K+ concentrations measured in 4–10 animals on each of the diets. There were no significant differences in serum or urine Na+ concentrations among the three groups. Predictably, the serum and urine K+ concentrations differed between animals on the three diets. The differences in weight gain among the three dietary groups were not statistically significant by ANOVA.
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Effects of diet on flow-stimulated net K+ secretion. At a slow flow rate of 1 nl·min–1·mm–1, the rate of net K+ secretion (in pmol·min–1·mm–1) in CCDs isolated from LK animals did not differ from that measured in HK animals (Fig. 2A). An increase in tubular fluid flow rate to 5 nl·min–1·mm–1 elicited an almost threefold stimulation in the rate of net K+ secretion in CCDs isolated from HK animals. CCDs from LK animals failed to exhibit a flow response (P = not significant) (Fig. 2A).
Net Na+ absorption was measured in the same tubules as described directly above. As shown in Fig. 2B, at a flow rate of 1 nl·min–1·mm–1, net Na+ absorption was lower in LK compared with HK animals (P < 0.01), presumably reflecting a state of hypoaldosteronism associated with K+ depletion. After an increase in tubular flow rate, net Na+ absorption increased significantly in both HK and LK animals. Although an increase in net Na+ absorption would be expected to increase the driving force for K+ secretion, this was not observed. We have previously reported that an increase in luminal flow rate in the CCD leads to an increase in net Na+ absorption that is unaccompanied by an increase in the lumen negative transepithelial voltage (44, 46, 62, 64). We have speculated in the past (46) that an increase in luminal flow rate enhances the paracellular permeability to Cl–, leading to movement of negative charge out of the lumen.
In two experiments using CCDs obtained from HK animals, luminal addition of 50 nM iberiotoxin inhibited flow-stimulated net K+ secretion (from 18.7 to 4.9 pmol·min–1·mm–1 and 15.6 to 4.1 pmol·min–1·mm–1) but was without consistent effect on net Na+ absorption (from 92.2 to 116.2 pmol·min–1·mm–1 and from 80.0 to 57.8 pmol·min–1·mm–1). These results, identical to those reported by us previously (64), are consistent with the notion that flow-stimulated K+ secretion is mediated by an iberiotoxin-sensitive maxi-K channel.
Effect of diet on expression of maxi-K channel subunit mRNA. The relative abundance of maxi-K channel - and 1–4-subunit transcripts was examined by real-time PCR analysis of whole kidney from LK and HK rabbits (Fig. 3) and single CCDs isolated from CK, LK, and HK rabbits (Fig. 4). All of the maxi-K channel subunits examined were expressed in whole kidney, and there were no significant differences in message levels in kidneys isolated from LK and HK animals (Fig. 3). Single CCDs expressed maxi-K - and 2–4-subunit mRNAs. However, 1 message was not detected in any -actin-positive CCD (Fig. 4), although this transcript was expressed in whole kidney (Fig. 3). Maxi-K - and 2–4 mRNA expression in CCDs isolated from HK animals was significantly greater than that detected in CK animals (P < 0.05) (Fig. 4). Conversely, maxi-K - and 2–4-subunit mRNA expression in CCDs isolated from LK animals was significantly less than that detected in CK animals (P < 0.05) (Fig. 4)
To confirm that the HK diet-induced increase in expression of maxi-K transcripts tested was specific to this channel and not due to a general stimulatory effect of K+ on transcription, the effect of dietary K+ intake on HKAc and HKAg -subunit transcript abundance was also determined. HKAc was markedly stimulated in CCDs from LK animals, whereas HKAg was significantly reduced in this experimental group (P < 0.05) (Fig. 5). The HK diet had no effect on expression of either HKA compared with the levels of expression detected in CK animals.
Western analysis of maxi-K channel protein in kidney homogenate. An immunoblot of whole rabbit kidney lysates prepared from CK, LK, and HK rabbits (n = 3 kidneys for each dietary group) was performed to discern whether dietary K+ intake led to changes in protein abundance at the level of the whole kidney (Fig. 6). Polypeptides with apparent molecular masses of 90 kDa were recognized by the antibody but not by antibody preincubated with the immunizing peptide (64). There was no apparent difference in abundance of whole kidney maxi-K expression among the three groups.
Effect of diet on immunolocalization of maxi-K channel -subunit. Given our concern that 1) diet-induced alterations in maxi-K channel expression in a subset of nephron segments, which represents only a minor fraction of the total renal mass, might be missed by Western blotting of whole kidney and 2) these studies do not address the cellular localization of channels, we sought to determine whether maxi-K channel -subunit expression and localization in single CCDs are regulated by diet. To examine this possibility, cryosections of rabbit kidneys were labeled with the antibody directed against the -subunit of the maxi-K channel and the principal cell-specific apical marker DBA and examined by indirect immunofluorescence microscopy.
In animals fed an HK diet, immunodetectable -subunit was detected in a linear pattern along the apical membrane (Figs. 7–9). Apical membrane maxi-K -subunit expression was limited primarily to -intercalated cells (Figs. 7–9), as the channel was present in cells expressing immunodetectable H+-ATPase (Fig. 8) and was only occasionally observed in cells that were labeled with peanut agglutinin, a -intercalated cell marker (Fig. 9). Apical labeling for -subunit was rarely observed in CCDs from animals fed a LK diet (Fig. 7). In the latter and CK animals, the distribution of immunodetectable channel protein appeared to be more diffuse and detected in a predominantly intracellular distribution, compared with CCDs from animals maintained on the HK diet.
The numbers of DBA-positive and DBA-negative cells, defined as principal and intercalated cells, respectively, and the number of DBA-negative (intercalated) cells showing linear apical maxi-K -subunit localization were counted in individual tubular profiles. Linear apical channel localization, not observed in DBA-positive cells, was present in 9% of intercalated cells in CK kidney sections and 29% of intercalated cells in HK animals (Table 3) (n 3 kidneys for each dietary group).
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DISCUSSION
The results of the present study indicate that dietary K+ intake regulates the capacity of the rabbit CCD for flow-stimulated K+ secretion, a transport process our group (62, 64) and others (2, 25) have proposed to be mediated by the maxi-K channel. It is well established that net K+ secretion and the density of apical conducting SK/ROMK channels in the CCD are exquisitely regulated in response to physiological demands, as reviewed in this discussion. The present study is, to our knowledge, the first to identify a contribution of the maxi-K channel to renal K+ adaptation in the mammalian CCD, a response that includes changes in steady-state abundance of maxi-K channel - and 2–4-subunit mRNAs (Fig. 4) as well as cellular localization of channel proteins in the CCD (Fig. 7). The increase in surface expression of maxi-K channels that we observed in response to an increase in dietary K+ intake is consistent with an increase in whole cell expression of maxi-K channels and/or a redistribution of channels from an intracellular pool to the plasma membrane.
Wang et al. (56) reported that COOH-terminal splice variants of maxi-K -subunit exhibit differences in cellular localization. Although our study does not address potential mechanisms that lead to a redistribution of K+ channels from an intracellular pool to the plasma membrane in response to an increase in dietary K+, it is possible that alternative splicing of the COOH terminus of maxi-K -subunit may contribute to the differences in cellular localization of maxi-K channel that we observed in animals maintained on different K+ diets (Fig. 7). The anti-maxi-K channel antibody that we used is directed against a conserved COOH-terminal domain of the -subunit that is present in -subunits cloned from rabbit kidney (GenBank no. BAA23747), as well as -subunits isolated from mouse, human, and dog. We are aware that this epitope resides in a region where alternative splicing may result in loss of this sequence, and thus not all splice variants may be detected with this antibody.
Cumulative evidence suggests that K+ adaptation in the rat is due, at least in part, to an increase in density of conducting SK channels in the apical membrane of the CCD (36, 37, 57, 58). The observation that the number of SK channels is not altered in CCDs isolated from rats on a low-Na+ diet, a maneuver that increases the circulating levels of aldosterone, suggests that the effect of HK intake on SK number is not mediated by this hormone (36). Furthermore, Frindt et al. (11) reported that an HK diet does not increase transcription of SK channels, since the relative amounts of ROMK mRNA were the same in single CCDs isolated from animals on HK and CK diets. The latter investigators thus proposed that the effect of an HK diet on the number of SK channels in rat CCD is achieved by a posttranslational modulation of a preexisting pool of channels or closely associated proteins (11). It should be noted that, in rabbits subjected to K+ loading, baseline net K+ secretion measured at the slow flow rate of 1 nl·min–1·mm–1, presumably mediated by the SK channel, was not statistically different from that observed in tubules isolated from LK animals (Fig. 2A). Whether this reflects a limited capacity of the rabbit vs. the rat to augment SK channel expression at the apical membrane of the CCD remains to be determined.
Controversy exists as to whether chronic K+ restriction decreases the number of conducting SK channels in the apical membrane of the rat CCD (37, 57). However, 2–5 days of LK intake has been shown to decrease the abundance of ROMK in both cortex and medulla (30) and specifically in the CCD, presumably due to an increase in endocytosis and degradation of the channel (5). Conservation of K+ and K+ reabsorption by the CCD may be further facilitated in the face of K+ restriction by upregulation of renal HKA, specifically at the apical membrane of the collecting duct (48). Both HKAg and HKAc transporters are expressed in the kidney but are differentially regulated (reviewed in Ref. 48). HKAc mRNA is upregulated in cortex of K+-depleted rats (32), whereas HKAg mRNA and protein abundance remain essentially unchanged in kidney under these same conditions (23). In the present study, we confirm, at the level of the single CCD, that K+ depletion induces mRNA encoding the -subunit of HKAc, which presumably facilitates K+ absorption from the tubular fluid (Fig. 5).
The results of the present study reveal that a fivefold increase in flow rate stimulated net K+ secretion only in CCDs isolated from HK but not LK animals (Fig. 2). The adaptation of the rabbit CCD to the HK diet is consistent with an increase in the apical K+ conductance and, based on studies in rats, likely reflects, at least in part, an increase in the number of active SK channels on the apical membrane. However, the dietary K+-induced increase in expression of mRNA encoding maxi-K - and 2–4-subunits, apical immunodetectable maxi-K -subunit in the CCD, and the sensitivity of the flow-induced increase in net K+ secretion to iberiotoxin provide compelling evidence for a major contribution of the maxi-K channel to the K+-adaptive response of the rabbit CCD.
Plasma aldosterone levels were not measured in the present study. However, on the basis of measurements from studies by others (61) in which rabbits were fed HK and LK diets within ranges of those utilized in the present study, it seems reasonable to assume that plasma aldosterone concentrations were greater in animals ingesting the HK compared with LK diets. In fact, we consider that low circulating levels of aldosterone in the LK rabbits accounts for the blunted rates of net Na+ absorption in CCDs isolated from this experimental group (Fig. 2). Low plasma concentrations of aldosterone are associated with undetectable apical ENaC activity in principal cells, as measured by patch-clamp analysis (34), and absence of immunodetectable ENaC subunits on the apical membrane of cells lining the aldosterone-sensitive distal nephron (24).
Conversely, the stimulation of net K+ secretion in HK animals (Fig. 2) may also be explained by an aldosterone-induced increase in the apical expression of conducting Na+ channels (24, 34) and net Na+ absorption (47), leading to an enhanced electrochemical diving force for K+ secretion into the urinary fluid. Aldosterone may also activate other intracellular second messenger systems that regulate maxi-K channel expression and activity. Aldosterone-induced stimulation of electrogenic Na+ transport in A6 renal cells may be dependent on a Ca2+/calmodulin-dependent protein kinase, as suggested by the sensitivity of the aldosterone-induced short-circuit current to W-7 and trifluoperazine (38). Ca2+/calmodulin-dependent protein kinase IV signaling has been implicated as pivotal in the regulation of alternative splicing of maxi-K pre-mRNAs through a defined RNA regulatory element, the Ca2+/calmodulin-dependent protein kinase IV-responsive RNA element (67). These observations suggest that Ca2+/calmodulin-dependent protein kinase may link increases in intracellular Ca2+ concentrations to transcriptional regulation of genes. Finally, CCD maxi-K channel activity is highly pH sensitive at physiological low cytosolic Ca2+ activities (16). To the extent that hyperaldosteronism is associated with metabolic alkalosis (12), the acid-base disturbance may have a direct causal role in increasing apical K+ channel activity during dietary K+ loading.
Patch-clamp analysis indicates that K+ loading stimulates maxi-K channel activity in rat distal colon (4). The effects of dietary K+ loading on maxi-K channel expression have not been rigorously examined in the mammalian CCD. However, long-term exposure (>7 days) of the amphibian Ambystoma tigrinum collecting duct to elevated environmental K+ increases their rate of renal K+ excretion (50). This adaptation is due, in part, to increases in apical maxi-K (10-fold) and ENaC (4-fold) channel density in collecting ducts isolated from these animals (53). Whereas these segments do not routinely exhibit maxi-K channel activity when studied at their resting membrane potential in 4 mM peritubular K+, collecting ducts bathed with a 15 mM K+ solution exhibit a high density of conducting maxi-K channels (52).
The present study is the first to characterize the identity of maxi-K -subunit expression in the mammalian CCD. The presence of 1 message in CNT (41) and its absence in all CCD samples studied in the present investigation suggest that -subunit expression is differentially regulated along the nephron. We now show that mRNA encoding 2–4 is present in the CCD and that the steady-state abundance of each subunit transcript is increased in response to dietary K+ loading when compared with levels in CCDs from animals on a CK diet. We acknowledge that our data do not address whether 2–4 protein is translated and expressed with the -subunit as a functional channel. Although single cells may contain multiple -subunit variants (19, 33, 42), our analysis of native tubules of heterogeneous cellular composition does not allow us to draw any conclusions about whether multiple -subunits are expressed in single cells.
The tissue-specific expression of -subunits has been proposed to allow for the assembly of a large number of distinct maxi-K channels in vivo, contributing to the functional diversity of native BK currents. In heterologous systems, coexpression of - with -subunits alters the voltage, Ca2+, and inhibitor sensitivity of the channel compared with expression of the -subunit alone. Specifically, coexpression of 1 with increases Ca2+ sensitivity, slows gating kinetics, and alters pharmacological properties of the channel (7, 20, 27). Coexpression of 2 with results in rapid and complete inactivation of channel currents (54, 55). The 3 partially inactivates maxi-K current and induces inward rectification of the current-voltage relationship (3, 54, 66); 4 increases the sensitivity for voltage, alters the gating behavior of the expressed channels in a Ca2+-dependent manner, and, if glycosylated, dramatically reduces iberiotoxin association rates (3, 15, 18, 28). On the basis of the inhibitory nature of the 2- and 3-subunits, it is unlikely that these subunits contribute significantly to the maxi-K conductance in native CCD. Whether the native channel is comprised of an -subunit alone, an -subunit associated with a nonglycosylated (iberiotoxin-sensitive) 4-subunit (18), or an as yet undescribed -subunit has yet to be determined.
The results of the present investigation do not allow us to discern whether the increases in dietary K+ load, plasma and/or cellular K+ concentration, and/or levels of circulating aldosterone stimulate de novo synthesis of new K+ channel protein(s) or the recruitment of preexisting K+ channels from within the cytosol to the apical membrane.
To begin to address this question, future efforts will need to investigate whether dietary Na+ depletion, which induces an even greater degree of secondary hyperaldosteronism than does K+ loading, elicits similar increases in maxi-K channel message, protein localization, and net K+ secretion compared with results reported in the present study.
In summary, our results provide compelling evidence to support the hypothesis that K+ adaptation to dietary K+ loading is associated with an increase in apical maxi-K channel activity in the mammalian CCD. Specifically, we now report that an increase in dietary K+ intake leads to increases in maxi-K channel - and 2–4-subunit mRNA expression and immunodetectable apical -subunit in the CCD. These results suggest that the adaptive response of the CCD to K+ loading includes transcriptional regulation of message and de novo synthesis of new maxi-K channel subunits, as well as the redistribution of channels from an intracellular pool to the plasma membrane, in sum leading to an increase in functional channel expression. We speculate that these adaptive responses, in concert with those already well described for the SK/ROMK channel, contribute to the maintenance of K+ homeostasis under conditions of K+ loading.
GRANTS
This work was supported by National Institutes of Health (NIH) Grants DK-038470 (to L. M. Satlin) and DK-051391 (to T. R. Kleyman). H. Zhou was supported by NIH Grant T32 HD-07537 (Training Grant in Developmental Biology of Membrane Transport; L. Satlin, principal investigator). T. Morimoto was supported by a Kidney and Urology Foundation of America Fellowship Grant.
Abstracts of this work were presented at the Annual Meetings of the American Society of Nephrology in 2003 (San Diego, CA) and 2004 (St. Louis, MO).
ACKNOWLEDGMENTS
The authors gratefully acknowledge Beth Zavilowitz for technical support and the MSSM Quantitative PCR Shared Research Facility.
FOOTNOTES
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.
F. Najjar and H. Zhou contributed equally to this work.
T. R. Kleyman and L. M. Satlin contributed equally to this work.
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