Colonic H+-K+-ATPase in K+ conservation and electrogenic Na+ absorption during Na+ restriction

Zachary Spicer1, Lane L. Clarke2, Lara R. Gawenis2, and Gary E. Shull1

1 Department of Molecular Genetics, Biochemistry and Microbiology, The University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524; and 2 Department of Biomedical Sciences, College of Veterinary Medicine and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Upregulation of the colonic H+-K+- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K+ conservation and electrogenic Na+ absorption in the colon when Na+-conserving mechanisms are activated. To test this hypothesis, wild-type (cHKA+/+) and cHKA-deficient (cHKA-/-) mice were fed Na+-replete and Na+-restricted diets and their responses were analyzed. In both genotypes, Na+ restriction led to reduced plasma Na+ and increased serum aldosterone, and mRNAs for the epithelial Na+ channel (ENaC) beta - and gamma -subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA-/- mice on a Na+-replete diet had elevated fecal K+ excretion. Dietary Na+ restriction led to increased K+ excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na+-replete or Na+-restricted diet. These results demonstrate that cHKA plays an important role in K+ conservation during dietary Na+ restriction and suggest that cHKA-mediated K+ recycling across the apical membrane is required for maximum electrogenic Na+ absorption.

Atp1al1; potassium absorption; sodium absorption; hydrogen secretion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE COLONIC H+-k+-atpase (cHKA), a member of the P-type family of ion transport ATPases that was cloned from distal colon (11), is expressed in apical membranes of distal colon surface cells (19, 27, 33). The existence of an H+-K+-ATPase in distal colon, or possibly two H+-K+-ATPases (1), was first indicated by physiological studies showing a linked pathway for H+ secretion and K+ absorption across the apical membrane of distal colon epithelial cells that was Na+ independent and sensitive to vanadate (12, 18, 23, 38, 39, 44). Expression studies using Xenopus oocytes demonstrated that cHKA is indeed an H+-K+-ATPase (6, 10), although there are data showing that the enzyme can substitute Na+ for H+ and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> for K+ (7-9, 32). The role of the pump in K+ homeostasis has been partially elucidated by using a gene-targeted mouse model. cHKA-/- mice exhibit a defect in colonic K+ conservation when fed either a control diet or a K+-depleted diet (30). Dietary K+ restriction does not affect cHKA mRNA, protein, or activity in colon (20, 35); however, in several models of hyperaldosteronism, including dietary Na+ restriction (35), treatment with aldosterone (20), and targeted mutation of the NHE3 Na+/H+ exchanger (36), cHKA mRNA is sharply induced.

In the rat distal colon, dietary Na+ restriction or treatment with aldosterone causes a switch in Na+ transport from predominantly electroneutral absorption via coupled Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange to electrogenic absorption via the amiloride-sensitive epithelial Na+ channel (16, 31). In addition, there is a sharp increase in both K+ secretion (16) and K+ absorption (14, 41). A K+-ATPase activity was suggested as the molecular mechanism of K+ absorption (41) and was hypothesized to recycle K+ secreted via the apical K+ channel (15, 16, 42). K+ absorption was inhibited by orthovanadate and ouabain (41), indicating that it is mediated by cHKA. cHKA mRNA was induced in distal colon of aldosterone- and dexamethasone-treated rats (20), and cHKA mRNA, protein, and activity increased in distal colon following dietary Na+ restriction (35).

The electrogenic absorption of Na+ from the lumen of the colon requires either the absorption of an anion or the secretion of a cation to maintain the appropriate electrochemical gradient across the apical membrane. For this reason, recycling of K+ that is secreted across the apical membrane may be critical for maximum electrogenic Na+ absorption via the epithelial Na+ channel (ENaC). Thus we hypothesized that the induction of cHKA during dietary Na+ restriction or other conditions in which aldosterone levels are elevated may allow an increased rate of K+ recycling across the apical membrane, thereby facilitating Na+ absorption. If so, then the loss of cHKA should impair both K+ conservation and electrogenic Na+ absorption. To test these hypotheses, we analyzed colonic Na+ and K+ conservation and electrogenic Na+ absorption in wild-type and cHKA-/- mice. The results demonstrate that the loss of cHKA impairs both K+ conservation and electrogenic Na+ absorption during dietary Na+ restriction.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Maintenance of mice and genotype analysis. Generation of cHKA-deficient mice was described previously (30). For the present study, the targeted null mutation was bred onto a C57BL/6 background. Mice were maintained in a pathogen-free barrier facility with free access to standard mouse chow and water until the time of the study.

Genotyping was performed by PCR analysis of genomic DNA isolated from tail biopsies. The PCR reaction used two cHKA gene-specific primers and one neomycin resistance gene-specific primer. The 5' cHKA primer (5'-CTGGAATGGACAGGCTCAACG-3') in conjunction with the 3' cHKA primer (5'-GTACCTGAAGAGCCCCTGCTG-3') amplified a 154-bp fragment of exon 20 from the wild-type allele. The 5' cHKA primer and the neomycin resistance gene primer (5'-CTGACTAGGGGAGGAGTAGAAGG-3') amplified a 298-bp fragment from the mutant allele that contains portions of exon 20 and the neomycin resistance gene. The PCR genotyping strategy and an example of the PCR amplifications are shown in Fig. 1.


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Fig. 1.   PCR genotyping strategy for colonic H+-K+-ATPase (cHKA)-deficient mice. A: part of the wild-type cHKA allele (top) and the mutant allele (bottom) with the neomycin resistance gene (neo) disrupting exon 20. Arrows indicate the relative positions of the primers used in a triplex PCR reaction that yielded 154- and 298-bp products for the wild-type and mutant alleles, respectively (see METHODS). B: agarose gel electrophoresis of ethidium bromide-stained PCR products from wild-type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice.

Western blot analysis. The distal colon was removed from two cHKA+/+ and two cHKA-/- mice and homogenized in 2 ml sucrose buffer [5 mM Tris, pH 7.5, 0.25 M sucrose, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. A 750-µl aliquot was centrifuged at 11,000 rpm for 30 min at 4°C. The supernatant was placed in a fresh tube and centrifuged at 50,000 rpm for 60 min at room temperature. The membrane pellet was resuspended in buffer B (50 mM HEPES, pH 7.6, 1 mM EDTA, and 1 mM PMSF). A portion of the membrane preparation (10 µl) was separated by electrophoresis in a 10% denaturing polyacrylamide gel and transferred to a nylon membrane. The primary antibody against cHKA (4) (a generous gift from Drs. Thomas DuBose and Juan Codina) was incubated with the membrane at a 1:1,000 dilution for 2 h. The secondary antibody was incubated at a 1:20,000 dilution for 1 h. Detection was performed with the SuperSignal kit (Pierce, Rockford, IL) according to the manufacturer's protocol. The protein on the membrane was then stained with Ponceau solution (Sigma) to confirm equal loading between lanes.

Dietary Na+ restriction studies. Studies were performed on age- and sex-matched adult cHKA+/+ and cHKA-/- mice. Mice were fed a control diet containing 1% NaCl, followed by a diet containing 0.01% NaCl (Harlan Teklad, Madison, WI), and urine and feces were collected daily. Urine volume was measured, and Na+ and K+ content were determined by flame photometry as described previously (30) and presented as 3-day averages. Feces from three consecutive days were pooled, weighed, dissolved in 0.75 N nitric acid, and centrifuged. Determination of fecal Na+ and K+ content was performed by flame photometry of the supernatant as described previously (30).

Determination of blood pH, gases, and electrolytes and serum aldosterone. Blood (50 µl) was collected from the tail vein of conscious mice and analyzed immediately for acid/base status, blood gases, and plasma electrolytes using a Chiron Diagnostics model 348 pH/blood gas analyzer (30). These analyses were performed at the end of both the control diet period and the Na+-restricted diet period. Serum aldosterone concentrations were also determined at the end of each period using a 125I radioimmunoassay as previously described (36).

Northern blot analyses. Total RNA from the kidneys of three animals in each experimental group and total RNA from proximal and distal colon of two animals in each group were isolated and pooled. Total RNA (10 µg) was denatured, separated by agarose gel electrophoresis, and transferred to a nylon membrane. Hybridization was performed as described previously (36) using cDNA probes for the alpha -subunit of cHKA; the beta 1-subunit of the Na+-K+-ATPase; channel-inducing factor (CHIF); the alpha -, beta -, and gamma -subunits of ENaC; and the mouse L32 ribosomal protein (as a loading control).

Bioelectric measurements. Transepithelial current measurements in the distal colon of cHKA+/+ and cHKA-/- mice were performed under short-circuit (Isc) conditions in Ussing chambers as described (3). Freshly isolated tissues were mounted in voltage-clamped Ussing chambers and were bathed in Krebs-Ringer-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution, pH 7.4 (in mM: 115 NaCl, 25 NaHCO3, 5 KCl, 1.2 MgCl2, 1.2 CaCl2, 10 serosal glucose, and 10 mucosal mannitol). In preliminary experiments, Isc was measured both before and after the sequential addition of 1 µM amiloride and then 50 µM amiloride to the luminal side of the tissue. Inhibition by 1 µM amiloride was only 54% of that observed with 50 µM amiloride. In subsequent experiments, Isc was measured both before and after the addition of 50 µM amiloride.

Statistics. Statistical significance was calculated by single-factor ANOVA, ANOVA-protected Bonferroni's t-test, or paired t-test as appropriate. Data are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary Na+ restriction was carried out using wild-type and cHKA-/- mice, which were housed individually in metabolic cages. The mice received food containing 1% NaCl during the 6-day control period (days 1-6) and 0.01% NaCl during the 12-day period of Na+ restriction (days 7-18). Urine and feces were collected and analyzed, and at the conclusion of each dietary period, blood was collected for analysis of blood gases, pH, plasma electrolytes, and serum aldosterone. Northern blot analyses of kidney and intestinal segments of Na+-replete and Na+-restricted mice were performed, and a separate group of Na+-replete and Na+-restricted mice were used for analysis of Isc in distal colon.

Body weights. At the beginning of the study, cHKA+/+ and cHKA-/- mice had similar body weights (23.8 ± 1.5 and 23.2 ± 1.1 g, respectively). The mice were also weighed at the end of the control diet period (day 6: cHKA+/+, 23.2 ± 1.1 g; cHKA-/-, 22.7 ± 1.1 g), at the midpoint of the Na+-restricted period (day 12: cHKA+/+, 23.8 ± 1.3 g; cHKA-/-, 22.7 ± 1.1 g), and at the end of the experiment (day 18: cHKA+/+, 24.4 ± 1.4 g; cHKA-/-, 23.3 ± 1.1 g). There were no significant differences in body weight for either genotype during the 18-day study, nor were there any significant differences in body weight between genotypes at any time point.

Western blot analysis of cHKA protein in distal colon of wild-type and cHKA-/-+mice. To confirm the effectiveness of the gene-targeting strategy, membranes were isolated from the distal colons of Na+-restricted wild-type and cHKA-/- mice and examined by Western blot analysis. An antibody directed against cHKA identified a ~110-kDa protein in cHKA+/+ distal colon, but only a trace band was seen in the same position in cHKA-/- distal colon (Fig. 2). The faint band seen in the knockout lanes may have been due to nonspecific hybridization, because it corresponds in position to a strong protein band observed in all lanes after staining with Ponceau solution (not shown). Alternatively, it may represent trace levels of a mutant protein, lacking some of the COOH terminal transmembrane domains, that could be produced from the aberrant cHKA transcripts.


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Fig. 2.   Western blot analysis of distal colon membranes from Na+-restricted cHKA+/+ and cHKA-/- mice. Membranes were isolated from distal colon of 2 cHKA+/+ and 2 cHKA-/- mice, separated by denaturing polyacrylamide gel electrophoresis, and probed with a cHKA-specific antibody. A single band, corresponding in size to that of the colonic H+-K+-ATPase, was clearly present only in the cHKA+/+ distal colon.

Urinary and fecal excretion of K+ and Na+. As shown in Fig. 3A, urinary K+ excretion was similar for Na+-replete cHKA+/+ (1,199 ± 113 µmol/3 days) and cHKA-/- (1,143 ± 89 µmol/3 days) mice but differed significantly following Na+ restriction (932 ± 88 and 754 ± 75 µmol/3 days, respectively; P < 0.01). Urinary Na+ excretion (Fig. 3B) was similar for cHKA+/+ and cHKA-/- mice during the control period (734 ± 122 and 855 ± 132 µmol/3 days, respectively) but decreased by ~99%, to 7.46 ± 0.85 and 6.96 ± 1.57 µmol/3 days, respectively, following Na+ restriction.


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Fig. 3.   Urinary K+ (A) and Na+ (B) excretion for cHKA+/+ and cHKA-/- mice. Animals were housed in metabolic cages and fed a 1% NaCl diet for 6 days (days 1-6), followed by a 0.01% NaCl diet for 12 days (days 7-18) (n = 9 mice for each genotype). Urine was collected, and K+ content and Na+ content were analyzed by flame photometry. Results are expressed as total K+ or Na+ excreted per 3 days. Values are means ± SE.

The quantity of feces excreted by cHKA+/+ and cHKA-/- mice did not differ significantly during the control (1.83 ± 0.06 and 2.11 ± 0.19 g/3 days, respectively) or Na+-restricted periods (2.21 ± 0.16 and 2.06 ± 0.13 g/3 days, respectively). As observed previously (30), fecal K+ excretion (Fig. 4A) was greater for cHKA-/- mice than for cHKA+/+ mice during the control diet period (379 ± 23 and 248 ± 16 µmol/3 days, respectively; P < 0.001). When fed a Na+-restricted diet, cHKA+/+ mice maintained the same level of fecal K+ excretion as during the control period. In contrast, during the last 3 days of Na+ restriction, fecal K+ excretion by cHKA-/- mice was 2.8-fold greater than that of wild-type mice (693 ± 38 and 247 ± 24 µmol/3 days, respectively; P < 0.001). Fecal excretion of Na+ (Fig. 4B) was similar for cHKA-/- and cHKA+/+ mice during the control period (249 ± 21 and 217 ± 19 µmol/3 days, respectively). When fed a Na+-restricted diet, fecal Na+ excretion decreased by >90% in both genotypes, although it was slightly greater in knockout than in wild-type mice (11.0 ± 0.5 and 13.7 ± 1.3 µmol/3 days, respectively; P = 0.023).


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Fig. 4.   Fecal K+ and Na+ excretion for cHKA+/+ and cHKA-/- mice. Animals were fed a 1% NaCl diet (days 1-6) followed by a 0.01% NaCl diet (days 7-18) (n = 13 for each genotype; this includes the 9 pairs in Fig. 3 and 4 additional pairs). Feces were pooled at 3-day intervals and were analyzed for K+ and Na+ content. A: total K+ excreted per 3 days for cHKA+/+ and cHKA-/- mice. All values for cHKA-/- mice are significantly greater (P < 0.001) than the corresponding values for cHKA+/+ mice. dagger P < 0.01 between K+ excreted on days 1-3 and K+ excreted on the indicated days for cHKA-/- mice only. B: total Na+ excreted per 3 days for cHKA+/+ and cHKA-/- mice. Values are means ± SE.

Plasma electrolytes and serum aldosterone. Plasma electrolytes and systemic acid/base status were analyzed at the end of each dietary period (Table 1). Serum Na+ concentrations in Na+-replete cHKA+/+ and cHKA-/- mice were 149.1 ± 0.5 mM and 150.0 ± 0.6 mM, respectively. Serum Na+ did not differ significantly between the two genotypes on either diet; however, both groups of mice became hyponatremic after 12 days of Na+ restriction, with serum Na+ of 146.8 ± 0.7 mM in wild-type mice (P = 0.034) and 146.9 ± 0.4 mM in the knockout (P < 0.01). Serum Cl- concentrations did not differ significantly between cHKA+/+ and cHKA-/- mice on a Na+-replete diet. During Na+ restriction, serum Cl- did not change in the knockout mice but was slightly elevated in wild-type mice (P = 0.024; this is most likely a statistical anomaly). Serum K+, pH, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations were essentially the same in both genotypes and were unaffected by diet.

                              
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Table 1.   Serum electrolytes and acid-base status of cHKA+/+ and cHKA-/- mice on 1% and 0.01% NaCl diets

Serum aldosterone levels (Fig. 5) were similar in wild-type and cHKA-/- mice (1,460 ± 204 pg/ml and 1,112 ± 101 pg/ml, respectively) at the end of the control diet period. After 12 days of Na+ restriction, serum aldosterone increased approximately threefold in both cHKA+/+ and cHKA-/- mice, to 4,333 ± 312 pg/ml and 3,524 ± 597 pg/ml, respectively.


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Fig. 5.   Serum aldosterone concentrations for cHKA+/+ and cHKA-/- mice after being fed a diet containing 1% NaCl (Normal) for 6 days and then a diet containing 0.01% NaCl (Restricted) for 12 days (n = 6 wild-type and 5 knockout mice). Values are means ± SE. *P < 0.01 between normal and Na+-restricted diets within each genotype; there were no significant differences between the 2 genotypes.

Northern blot analysis of kidneys and colon. Northern blot hybridization was performed using kidney RNA from Na+-replete and Na+-restricted mice (Fig. 6, left). A previous study showed that cHKA mRNA is expressed at almost undetectable levels in kidneys of K+-replete wild-type mice but is sharply induced by K+-depletion (30). However, we observed no signal for cHKA mRNA in kidneys of Na+-replete mice of either genotype, and there was no evidence of cHKA induction in response to Na+ restriction. Na+ restriction had no apparent effect on kidney mRNA levels for the ENaC subunits, CHIF, or the Na+-K+-ATPase beta 1-subunit.


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Fig. 6.   Northern blot analysis of RNA from kidney, proximal colon, and distal colon of cHKA+/+ and cHKA-/- mice maintained on normal Na+-replete (N) or Na+-restricted (R) diets. For each genotype and dietary group, total RNA was isolated and pooled from the kidneys of 3 mice and from the proximal and distal colons of 2 mice. Each lane contained 10 µg of total RNA. The probes were: alpha -subunit of cHKA; beta 1 subunit of Na+-K+-ATPase (beta 1NKA); channel-inducing factor (CHIF); alpha -, beta -, and gamma -subunits of the epithelial Na+ channel (alpha ENaC, beta ENaC, and gamma ENaC); and the L32 ribosomal protein (L32) as a loading control.

Northern blot analyses of RNA from proximal and distal colon revealed similar changes in both genotypes in response to Na+ restriction (Fig. 6, right). cHKA mRNA was not detected in proximal colon of Na+-replete mice of either genotype, but very low levels could be detected after Na+ restriction (Fig. 6; longer exposures not shown). In distal colon, expression of both the wild-type cHKA mRNA in cHKA+/+ mice and the mutant mRNA (which is larger due to insertion of the neomycin resistance gene) in cHKA-/- mice were induced after Na+ restriction. Na+-K+-ATPase beta 1-subunit mRNA increased slightly in both proximal and distal colon of cHKA+/+ and cHKA-/- mice after Na+ restriction, whereas mRNA for the alpha -subunit of ENaC was unchanged. In Na+-replete mice, CHIF mRNA was not detected in proximal colon, and with the short (3 h) exposure time shown in Fig. 6, CHIF mRNA was detected at only trace levels in distal colon. In contrast, it was sharply induced in distal colon of both genotypes after Na+ restriction. Na+ restriction also caused a sharp induction of the mRNAs encoding the beta - and gamma -subunits of ENaC in proximal and distal colon of both groups of mice.

Isc in the distal colon of Na+-restricted cHKA+/+ and cHKA-/- mice. In an initial set of experiments designed to test the concentrations of amiloride needed to fully inhibit ENaC, we measured transepithelial current in the distal colon of Na+-restricted cHKA+/+ and cHKA-/- mice under short-circuit conditions in the absence of amiloride and then in the presence of 1 µM and then 50 µM amiloride. Dietary Na+ restriction was used to enhance the magnitude of the ENaC-mediated currents. The basal Isc in cHKA-/- colon was significantly lower than that in cHKA+/+ colon (-39.1 ± 6.7 µA/cm2 and -193.2 ± 14.1 µA/cm2, respectively; P < 0.01; Fig. 7). After the luminal addition of 1 µM amiloride, the Isc decreased in both groups (cHKA-/-, -13.7 ± 3.3 µA/cm2; cHKA+/+, -59.7 ± 6.9 µA/cm2; P < 0.01). Addition of 50 µM amiloride led to a further reduction in the Isc (cHKA-/-, 0.5 ± 1.2 µA/cm2; cHKA+/+, 22.7 ± 4.1 µA/cm2; P < 0.01). In subsequent experiments, 50 µM amiloride was used to inhibit the ENaC-mediated current.


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Fig. 7.   Effects of amiloride on short-circuit current (Isc) in the distal colon of Na+-restricted cHKA+/+ and cHKA-/- mice. Isc under basal conditions and after the sequential addition of 1 µM and 50 µM amiloride are shown (n = 6 cHKA+/+ and 5 cHKA-/- mice). Values are means ± SE. *P < 0.01 between cHKA+/+ and cHKA-/- within each treatment. dagger P < 0.01 for difference from basal current of same genotype.

In the second set of experiments, transepithelial currents were measured in the distal colon of both Na+-replete and Na+-restricted cHKA+/+ mice and cHKA-/- mice. The basal Isc did not differ significantly between Na+-replete cHKA-/- and cHKA+/+ mice (-22.9 ± 3.5 and -35.6 ± 7.6 µA/cm2, respectively); however, after the luminal addition of 50 µM amiloride, the Isc differed significantly between the two groups (cHKA-/-, -9.2 ± 1.6 µA/cm2; cHKA+/+, 5.9 ± 2.6 µA/cm2; P < 0.01) (Fig. 8A). The amiloride-sensitive Isc, representing the contribution from ENaC, was approximately threefold greater in Na+-replete wild-type distal colon than in that of the mutant (41.5 ± 7.2 and 13.7 ± 3.3 µA/cm2, respectively; P < 0.01; Fig. 8B). Compared with that observed under Na+-replete conditions, the basal Isc measured under conditions of dietary Na+ restriction increased ~4.9-fold in cHKA+/+ mice (to -173.0 ± 13.7 µA/cm2; P < 0.01) and increased only 2.2-fold in cHKA-/- mice (to -50.2 ± 7.7 µA/cm2; P < 0.01) (Fig. 8A). After the luminal addition of 50 µM amiloride, the Isc did not differ significantly between cHKA-/- and cHKA+/+ mice (-7.6 ± 3.5 and 8.4 ± 7.2 µA/cm2, respectively). The amiloride-sensitive Isc was ~4.3-fold greater in Na+-restricted cHKA+/+ distal colon than in that of the mutant (181.5 ± 14.5 and 42.6 ± 6.8 µA/cm2, respectively; P < 0.01; Fig. 8B).


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Fig. 8.   Isc in the distal colon of cHKA+/+ and cHKA-/- mice after being fed a 1% NaCl diet (normal) or a 0.01% NaCl diet (restricted) for 12 days. A: Isc under basal conditions and after the addition of amiloride. B: change in Isc after the addition of amiloride (n = 8 Na+-replete cHKA+/+ mice, 8 Na+-replete cHKA-/- mice, 12 Na+-restricted cHKA+/+ mice, and 11 Na+-restricted cHKA-/- mice; the Na+- restricted mice include the 6 cHKA+/+ and 5 cHKA-/- mice analyzed in Fig. 7). Values are means ± SE. *P < 0.01 between cHKA+/+ and cHKA-/-. dagger P < 0.01 between 1% NaCl diet and 0.01% NaCl diet.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies with cHKA-/- mice have shown that the cHKA plays an important role in K+ conservation in the colon during dietary K+ restriction (30), although its mRNA is not induced in the colon under these conditions (35). K+ restriction and the resulting low serum aldosterone levels cause a reduction in apical K+ secretion; hence, there is much less K+ to recover and normal levels of the pump are apparently sufficient to reduce fecal K+ losses to very low levels (30). In contrast, cHKA mRNA, protein, and activity in distal colon are increased in response to dietary Na+ deprivation or elevated serum aldosterone levels (20, 35), suggesting that its activity might be particularly important under conditions in which electrogenic Na+ absorption predominates in the colon (22). In the current study, we used cHKA knockout mice to examine the role of the pump in K+ conservation during dietary Na+ restriction and to determine whether its absence affects the rate of electrogenic Na+ absorption.

Dietary Na+ restriction or treatment with aldosterone has been shown to stimulate NaCl absorption in the rat proximal colon via coupled electroneutral Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (16). In the rat distal colon, however, aldosterone reduces electroneutral transport of Na+ and stimulates electrogenic transport via ENaC, the amiloride-sensitive Na+ channel (16). To maintain the membrane potential needed for electrogenic absorption of Na+, aldosterone stimulates secretion of K+ through a Ba2+-sensitive K+ channel (42). Thus it seemed likely that a major function of cHKA in the distal colon of Na+-restricted mice would be the conservation of K+ secreted through apical K+ channels; however, an additional function also seemed likely. Secretion of K+ and electrogenic Na+ absorption have been shown to be dependent on the Na+-K+-ATPase and Na+-K+-2Cl- cotransporter (42), both of which mediate K+ uptake across the basolateral membrane. Nevertheless, it was reasonable to anticipate that recycling of secreted K+ via the colonic H+-K+-ATPase, which would be expected to enhance the apical K+ gradient, might also be required for maximum electrogenic Na+ absorption.

Neither cHKA+/+ nor cHKA-/- mice fed a 0.01% NaCl diet exhibited overt pathology, morbidity, or loss of body weight. Serum K+, Cl-, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations and serum pH were not affected by Na+ restriction, but both genotypes exhibited a mild reduction in plasma Na+ and elevated serum aldosterone levels by the end of the Na+ restriction period. In the distal colon, Na+ restriction induced cHKA mRNA, and in both proximal and distal colon it caused a slight induction of mRNA for the Na+-K+-ATPase beta 1-subunit, which has been shown to associate with the cHKA alpha -subunit in vivo (4, 26). CHIF, which activates a K+ current (2), was induced only in distal colon. In both proximal and distal colon, ENaC mRNAs were expressed at similar levels in Na+-replete cHKA+/+ and cHKA-/- mice, and ENaC beta - and gamma -subunit mRNAs were sharply induced in both segments during Na+ restriction. These results demonstrated that dietary Na+ restriction in the mouse had the expected effects on Na+ homeostasis and serum aldosterone and that it modified ion transporter mRNA expression in cHKA+/+ and cHKA-/- colon in a manner similar to that reported for rat colon (2, 13, 20, 35, 43).

When fed a control diet containing 1% NaCl, the K+ content of the feces of cHKA-/- mice was ~50% higher than for cHKA+/+ mice. During the Na+ restriction period, wild-type mice maintained the same fecal K+ levels as observed on the Na+-replete diet, but the fecal K+ content of the knockout increased to a level 2.8-fold greater than that of wild-type mice. These data provide direct confirmation of the hypothesis that cHKA plays an important role in colonic K+ conservation not only during dietary K+ restriction, as shown previously (30), but also during dietary Na+ restriction. This suggests that part of the biological rationale for the induction of cHKA in colon by Na+ restriction or under conditions in which serum aldosterone is elevated, as observed previously in rat and mouse colon (20, 35, 36), is to recover the K+ being secreted under these conditions (16).

The Na+ content of the feces of both cHKA+/+ and cHKA-/- mice decreased by ~90% during the Na+ restriction period, although it remained slightly higher in cHKA-/- mice than in cHKA+/+ mice. To analyze Na+ transport in the distal colon of cHKA+/+ and cHKA-/- mice, Isc in colons of Na+-replete and Na+-restricted mice were analyzed. These experiments showed that the amiloride-sensitive Na+ current in both the Na+-replete and Na+-restricted cHKA-/- colon was substantially less than that observed in the corresponding cHKA+/+ colon. Although ENaC activity was induced by Na+ depletion in both genotypes, the Na+ current present in Na+-restricted cHKA-/- colon was essentially the same as that observed in Na+-replete cHKA+/+ colon and ~25% of that in Na+-restricted cHKA+/+ colon (see Fig. 8). But why would the loss of cHKA cause a reduction in electrogenic Na+ absorption? One possibility, as suggested by others (22), is that cHKA functions as a K+-recycling mechanism to maintain K+ secretion and a favorable membrane potential for electrogenic Na+ absorption. Similar ion recycling mechanisms have been observed in other systems. For example, gene knockout studies showed that a reduction in the levels of the Na+-K+-ATPase alpha 2-isoform, but not the alpha 1-isoform, enhances cardiac contractility and Ca2+ loading of cardiac myocytes (20). It was concluded that regulation of subsarcolemmal Na+ concentrations by the alpha 2-isoform controls the activity of the Na+/Ca2+ exchanger, with the resulting alteration in intracellular Ca2+ affecting cardiac contractility (21). In another recent example, the extrusion of H+ via an apical Na+/H+ exchanger was shown to reduce the subapical H+ concentration in colonocytes, thereby creating a pH microclimate that stimulated the uptake of short-chain fatty acids (17). Finally, it is well established that recycling of K+ by the renal outer medullary K+ channel is critical for activity of the apical Na+-K+-2Cl- cotransporter of the thick ascending limb and for accompanying paracellular transport of Na+ (37). The results of the present study indicate that K+ absorption by the colonic H+-K+-ATPase reduces luminal K+ concentrations and are consistent with the possibility that it serves as a K+-loading mechanism that increases subapical K+ concentrations. If this hypothesis is correct, then the resulting transmembrane K+ gradient would enhance electrogenic K+ secretion, thereby contributing to the electrical potential needed for maximum electrogenic Na+ absorption.

Given that ENaC-mediated Na+ currents in cHKA-/- distal colon were much less than in wild-type distal colon, it was surprising that cHKA-/- mice exhibited only a mild increase in fecal Na+ excretion, even when maintained on a Na+-depleted diet. A possible explanation for this discrepancy is that apical Na+/H+ exchange activity is higher in cHKA-/- distal colon than in wild-type distal colon, thereby compensating for the reduction in ENaC activity. Our measurements of ENaC activity do not allow an estimation of the relative amounts of Na+ being absorbed via the electroneutral process of coupled Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, electrogenic transport via ENaC, or other processes that have not yet been well characterized. Similarly, the relative amounts of K+ being absorbed by cHKA or other K+-recovery mechanisms, such as the putative second H+-K+-ATPase (1), is unclear. An additional uncertainty is the physiological function of the apparent Na+ and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> ATPase and transport activity that has been attributed to cHKA or the second putative H+-K+-ATPase (7-9, 32). In the studies described here, we have considered only the functions of cHKA operating in an H+/K+ exchange mode. In future studies, it will be important to develop a quantitative understanding of the relative contributions of the various apical transport mechanisms and to understand the physiological functions and mechanistic basis for the putative Na+/K+ and H+/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> exchange modes of the colonic H+-K+-ATPase (7-9). Interestingly, CHIF is related to the Na+-K+-ATPase gamma -subunit (40), raising the intriguing possibility that it might interact with and affect the activity of cHKA. The conditions under which CHIF is induced, such as during dietary Na+ depletion or in diarrheal states such as in the NHE3-deficient mouse, are those in which the H+/K+ exchange mode would likely play an important role in the recovery HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+. As far as we are aware, the possible interactions of CHIF with cHKA and the effects of such an interaction on its ion dependence have not been examined

Although our primary objective was to investigate the role of cHKA in K+ conservation and electrogenic Na+ absorption in the colon, we were also interested in determining whether dietary Na+ restriction might affect renal Na+ handling. cHKA is expressed at very low levels in rat kidney under K+-replete conditions (11), but its mRNA and protein are induced by K+ deprivation (3, 25), a condition in which aldosterone levels are low. Although cHKA mRNA is sharply induced in mouse kidney by K+ deprivation (30), it was not induced by Na+ deprivation, nor was there any significant increase in urinary Na+ excretion in the knockout. Our results are consistent with previous experiments showing that aldosterone does not induce cHKA mRNA in kidney, as it does in colon (20). These data suggest that cHKA is not required for Na+ conservation in the mouse kidney during dietary Na+ restriction.

As illustrated in the model shown in Fig. 9, the current and previous studies of cHKA suggest that it carries out, in concert with other transporters, at least two important physiological functions in the colon. A role in K+ conservation was clearly demonstrated by the earlier observation that cHKA-/- mice exhibit greater fecal K+ excretion and become more hypokalemic than wild-type mice when maintained on a K+-deficient diet (30), and the results of the present study show that cHKA is also important for K+ conservation during dietary Na+ restriction. Depending on whether or not it is coupled with the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, which appears to be the product of the chloride diarrhea gene (CLD; also referred to as DRA) (24, 29), cHKA may contribute to either KCl or KHCO3 absorption. In addition to K+ conservation, cHKA also appears to contribute, as part of a coupled system, to electrogenic Na+ absorption. Under Na+-replete conditions, NaCl absorption is thought to be mediated primarily by the coupled activities of the NHE3 Na+/H+ exchanger and the CLD Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (24, 29, 36); the function of cHKA under these conditions appears to be largely restricted to K+ conservation, although an impairment of electrogenic Na+ absorption is apparent even under these conditions. Our data suggest, however, that K+ recycling by cHKA is required for a high rate of electrogenic Na+ absorption during dietary Na+ restriction. The concerted activities of ENaC, the apical K+ channel(s), and cHKA, which are all present in the apical membrane of surface cells (19, 28, 32), would be functionally equivalent to Na+/H+ exchange, at least when cHKA functions in an H+/K+ exchange mode. With varying degrees of coupling between these transporters and the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, this multicomponent system may contribute to the recovery of Na+, K+, Cl-, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> under a variety of physiological conditions.


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Fig. 9.   Proposed model of transport mechanisms mediating transepithelial KCl and NaCl absorption in colonic surface cells. KCl absorption occurs by coupled Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+/K+ exchange (via cHKA) on the apical membrane and by KCl cotransport (34) and possibly K+ and Cl- channel activity on the basolateral membrane. Under Na+-replete conditions, NaCl absorption occurs largely via coupled Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, whereas during dietary Na+ restriction Na+ is absorbed electrogenically via ENaC (16, 31). The appropriate electrochemical gradients for Na+ absorption are maintained by K+ secretion via an apical membrane channel (14, 42), K+ recycling and H+ secretion by cHKA (38, 39, 41), and the activities of the Na+-K+-ATPase and Na+-K+-2Cl- cotransporter (42) on the basolateral membrane.


    ACKNOWLEDGEMENTS

We thank Drs. Thomas DuBose and Juan Codina for the generous gift of the cHKA antibody used in this study.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50594 and DK-48816.

Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry and Microbiology, Univ. of Cincinnati, College of Medicine, 231 Albert Sabin Way, ML 524, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.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.

Received 5 March 2001; accepted in final form 3 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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