Am J Physiol Renal Physiol 310: F1035–F1046, 2016.
First published March 9, 2016; doi:10.1152/ajprenal.00010.2016.
Hyperaldosteronism after decreased renal K⫹ excretion in KCNMB2
knockout mice
Casper K. Larsen,1 Iben S. Jensen,1 Mads V. Sorensen,1,2 Pauline I. de Bruijn,1 Markus Bleich,3
Helle A. Praetorius,1 and Jens Leipziger1
1
Department of Biomedicine, Physiology, and Health, Aarhus University, Aarhus, Denmark; 2Aarhus Institute for Advanced
Studies, Aarhus University, Aarhus, Denmark; and 3Institute of Physiology, Christian-Albrechts-University, Kiel, Germany
Submitted 6 January 2016; accepted in final form 8 March 2016
KCa1.1 channel; potassium excretion; ␤2-subunit; ␣-subunit of the
KCa1.1 channel
excrete excess dietary K⫹ via active distal tubular
K⫹ secretion (16, 55). The principal cells of the connecting
tubule (CNT) and cortical collecting duct (CCD) are the main
site of renal outer medullary K⫹ (ROMK) channel-mediated
K⫹ secretion, driven by Na⫹ influx via epithelial Na⫹ channels
(ENaC) (28, 30, 49). In 2001, an additional K⫹ secretory
mechanism, which could be stimulated by high tubular flow
rates, was demonstrated (51). Inhibition of flow-induced K⫹
secretion (FIKS) by charybdotoxin or tetraethylammonium
indicated that Ca2⫹-activated K⫹ (KCa)1.1 channels were reTHE KIDNEYS
Address for reprint requests and other correspondence: J. Leipziger, Dept. of
Biomedicine, Physiology, and Health, Aarhus Univ., Ole Worms Allé 3, Bld.
1160, Aarhus C 8000, Denmark (e-mail: [email protected]).
http://www.ajprenal.org
sponsible for FIKS, and this was subsequently confirmed by
inhibition of FIKS with the specific KCa1.1 inhibitor iberiotoxin (52). A functional role of KCa1.1 channels in distal
tubular K⫹ secretion was further strengthened by a micropuncture study (3), where an iberiotoxin-sensitive K⫹ secretion was
detected in distal tubules from both ROMK⫹/⫹ and ROMK⫺/⫺
mice. Thus, the current understanding is that two secretory K⫹
channels are responsible for renal distal tubular K⫹ secretion (41).
ROMK-mediated K⫹ secretion is regarded as the primary
route for K⫹ secretion under normal dietary K⫹ intake (13),
but, interestingly, KCa1.1 channels contribute to K⫹ secretion
in mice on a normal K⫹ diet (3). Both ROMK (15, 48) and
KCa1.1 (31, 38) channels are upregulated in animals challenged
with high-K⫹ diets, indicative of the need for both channels
when renal K⫹ secretion must be increased.
The KCa1.1 channel is composed of four pore-forming
␣-subunits and four modulatory ␤-subunits, of which there are
four isoforms (␤1–␤4) (33). Recently, a novel group of four
KCa1.1 ␥-subunits (␥1–␥4) were identified, which modulate the
channel similarly to the ␤-subunits (54). KCa1.1 channels are
activated by membrane depolarization and increased intracellular Ca2⫹ concentration, and ␤- or ␥-subunits increase the
Ca2⫹ sensitivity of the channel (25, 32, 53, 54). KCa1.1
activity, protein, and mRNA have been detected in renal
epithelia in the CNT and CCD (11, 31, 34, 36, 50, 52). KCa1.1
channels have been detected in the apical membrane of both
principal and intercalated cells by patch clamp; however, their
density was higher in intercalated cells (34, 36). Localization
of KCa1.1 channels primarily in intercalated cells is supported
by immunohistochemistry in both rabbits (11, 52) and mice
(50), although they are also detectable in principal cells of
murine medullary collecting ducts (MCDs) (50). mRNA expression of ␥-subunits in human kidneys is very low (54),
whereas mRNA and protein expression of ␤1-, ␤2-, and ␤4subunits has been shown in whole mouse kidneys (18), and
mRNA for ␤2-, ␤3-, and ␤4-subunits was detected in rabbit
CCDs (11, 31).
The importance KCa1.1 for renal K⫹ excretion has been
studied in global knockout mice of the ␣-subunit (KCNMA1⫺/⫺
mice), ␤1-subunit (KCNMB1⫺/⫺ mice), and ␤4-subunit
(KCNMB4⫺/⫺ mice) of KCa1.1. KCNMA1⫺/⫺ mice do not
exhibit FIKS, which is observed in wild-type mice in response
to increased tubular flow (38). Despite the lack of KCa1.1mediated K⫹ secretion, KCNMA1⫺/⫺ mice are normokalemic,
even on a high-K⫹ diet. Protein expression of the ␤1-subunit
has been demonstrated specifically in the apical membrane
region of all cells in the CNT (37), and functional data suggest
reduced urinary K⫹ excretion in this mouse (19).
KCNMB4⫺/⫺ mice show no K⫹ handling phenotype on a normal
1931-857X/16 Copyright © 2016 the American Physiological Society
F1035
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Larsen CK, Jensen IS, Sorensen MV, de Bruijn PI, Bleich M,
Praetorius HA, Leipziger J. Hyperaldosteronism after decreased
renal K⫹ excretion in KCNMB2 knockout mice. Am J Physiol Renal
Physiol 310: F1035–F1046, 2016. First published March 9, 2016;
doi:10.1152/ajprenal.00010.2016.—The kidney is the primary organ
ensuring K⫹ homeostasis. K⫹ is secreted into the urine in the distal
tubule by two mechanisms: by the renal outer medullary K⫹ channel
(Kir1.1) and by the Ca2⫹-activated K⫹ channel (KCa1.1). Here, we
report a novel knockout mouse of the ␤2-subunit of the KCa1.1
channel (KCNMB2), which displays hyperaldosteronism after decreased renal K⫹ excretion. KCNMB2⫺/⫺ mice displayed hyperaldosteronism, normal plasma K⫹ concentration, and produced dilute
urine with decreased K⫹ concentration. The normokalemia indicated
that hyperaldosteronism did not result from primary aldosteronism.
Activation of the renin-angiotensin-aldosterone system was also ruled
out as renal renin mRNA expression was reduced in KCNMB2⫺/⫺
mice. Renal K⫹ excretion rates were similar in the two genotypes;
however, KCNMB2⫺/⫺ mice required elevated plasma aldosterone to
achieve K⫹ balance. Blockade of the mineralocorticoid receptor with
eplerenone triggered mild hyperkalemia and unmasked reduced renal
K⫹ excretion in KCNMB2⫺/⫺ mice. Knockout mice for the ␣-subunit
of the KCa1.1 channel (KCNMA1⫺/⫺ mice) have hyperaldosteronism,
are hypertensive, and lack flow-induced K⫹ secretion. KCNMB2⫺/⫺
mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1⫺/⫺ mice but were normotensive and displayed
intact flow-induced K⫹ secretion. Despite elevated plasma aldosterone, KNCMB2⫺/⫺ mice did not display salt-sensitive hypertension
and were able to decrease plasma aldosterone on a high-Na⫹ diet,
although plasma aldosterone remained elevated in KCNMB2⫺/⫺
mice. In summary, KCNMB2⫺/⫺ mice have a reduced ability to
excrete K⫹ into the urine but achieve K⫹ balance through an aldosterone-mediated, ␤2-independent mechanism. The phenotype of
KCNMB2 mice was similar but milder than the phenotype of
KCNMA1⫺/⫺ mice.
DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
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MATERIALS AND METHODS
Mice, diets, and housing. The KCNMB2 strain was generated in the
129/SvEvBrd ⫻ C57BL/6 background by the Texas Institute of
Genomic Medicine (College Station, TX) by truncation of exon 3 and
deletion of exons 4 and 5. Mice were bred from heterozygous
breeding families to yield KCNMB2⫹/⫹ and KCNMB2⫺/⫺ littermates. Genotyping was performed with the following primers: forward 5=-GCTAACCCCAGTGTGTCCT-3=, reverse 2: 5=- GTGATGGACCGTACTCTCCA-3=, and LacZrev 5=- GCGGATTGACCGTAATGGGATAGG-3=. Mice were kept in cages with no more than 4
mice/cage and given access to food and water ad libitum. A normal
diet (Altromin 1310, K⫹ content: 10 g/kg and Na⫹ content: 2 g/kg)
was given unless otherwise stated. For some experiments, mice were
fed the normal diet supplemented with KCl to yield a dietary K⫹
content of 50 g/kg, normal diet supplemented with NaCl to yield a
dietary Na⫹ content of 32g/kg, or an aldosterone-inducing high-K⫹/
low-Na⫹ diet (Altromin C1050, K⫹ content: 50 g/kg and Na⫹
content: 0.2 g/kg). Mice of either sex were used at an age between 6
and 16 wk. Experiments were performed in accordance with the
Danish legislation on the protection of animals (licence no. 2013-152934-00966).
Anesthesia. For most experiments, mice were anesthetized with an
intraperitoneal injection of a solution of ketamine (100 mg/kg body
wt) and xylazine (10 mg/kg body wt) in sterile water. For blood
sampling by decapitation, mice were anesthetized by isoflurane inhalation.
Semiquantitative RT-PCR. Mice were anaesthetized with ketaminexylazine and perfused with 20 ml PBS through the left ventricle.
Kidneys were harvested and decapsulated, whereupon mRNA was
isolated from whole kidneys using RNeasy Mini Kits (Qiagen). cDNA
was generated with Superscript III (Invitrogen) and Superase (Invitrogen). Semiquantitative RT-PCR analysis was performed with the
Taqman Gene Expression Assay (Applied Biosystems) against
Fig. 1. Appearance of isolated renal tubules
in phase-contrast microscopy. A: proximal
tubules (PTs). B: thick ascending limbs
(TALs). C: tubular segments composed of
distal convoluted tubules (DCTs) and connecting tubules (CNTs)/cortical collecting
ducts (CCDs). Branching points in connecting tubules were used as sorting criterium.
The transition from the DCT to the CNT was
visible as an abrupt change in tubular diameter and morphology (*). In the right tubular
segment, the transition from the TAL to the
DCT is apparent (§), thus providing the
length of a typical DCT segment. D: medullary collecting ducts (MCDs) with (left) or
without (right) the CNT/CCD segment attached. MCDs were cut from the CNT/CCD
at a length from the branching point equal to
the length of the CNT (arrow).
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diet but develop hyperkalemia when challenged with a high-K⫹
diet because of an apparently reduced renal K⫹ excretion (9, 22).
Here, we describe a knockout mouse of the ␤2-subunit
(KCNMB2⫺/⫺), which shows hyperaldosteronism secondary
to reduced renal K⫹ excretion. KCNMB2⫺/⫺ mice were
normokalemic but required elevated plasma aldosterone to
achieve K⫹ homeostasis. Blocking the mineralocorticoid receptor (MR) unmasked the renal K⫹ excretion deficiency and
induced mild hyperkalemia in KCNMB2⫺/⫺ mice. As opposed
to KCNMA1⫺/⫺ mice, KCNMB2⫺/⫺ mice displayed a milder
phenotype with intact FIKS. In patients with primary hyperaldosteronism, elevated plasma aldosterone results in the development of hypertension (20). In KCNMB2⫺/⫺ mice, however,
elevated plasma aldosterone did not lead to hypertension on
either a normal diet or on a high-Na⫹ diet. The aldosteronemediated compensatory mechanism in KCNMB2⫺/⫺ mice did
not include an apparent regulation of protein expression of the
renal Na⫹-Cl⫺ cotransporter (NCC) or the ␥-subunit of the
epithelial Na⫹ channel (␥-ENaC). These results further strengthen
the necessary role of the KCa1.1 channel in renal K⫹ excretion.
DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
Fig. 2. Efficacy of 4 days of eplerenone treatment. Mice with knockout of the
␤2-subunit of the KCa1.1 channel (KCNMB2⫹/⫺ mice) were fed the aldosterone-inducing high-K⫹/low-Na⫹ diet (HKLSD) for 24 h, which significantly
upregulated epithelial Na⫹ channel (ENaC) activity. Eplerenone treatment
abolished the upregulation of ENaC activity. KCNMB2⫹/⫺ mice treated with
eplerenone and fed the aldosterone-inducing diet for 24 h displayed no
upregulation of ENaC activity compared with KCNMB2⫹/⫺ mice fed a normal
diet. Data are individual measurements (n ⫽ 4 mice/group), means are
indicated by solid lines, and comparisons were made with an unpaired
Student’s t-test.
Fig. 3. mRNA expression of KCa1.1 channel subunits in the whole kidney from
KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice relative to an average of the reference
genes [␤-actin and hypoxanthine-guanine phosphoribosyltransferase (HPRT);
n ⫽ 6 for KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice]. Data are shown as means ⫾
SEM, and comparisons were made with an unpaired Student’s t-test. **P ⬍
0.001; ***P ⬍ 0.0001. §KCNMB3 and KCNMB4 were detectable, but
threshold cycle (Ct) values were too high (Ct ⬍ 38) to allow a comparison of
expression levels.
gathered per sample. In unpublished experiments by M. Bleich and N.
Himmerkus (University of Kiel, Kiel, Germany), tubules gathered at
this ratio produced comparable actin band densities in Western blots.
mRNA was isolated from tubule samples with RNeasy Micro Kits
Fig. 4. Representative gels of end-point RT-PCR for KCNMA1 and KCNMB2
in isolated renal tubules. Colonic crypts were used as positive control tissue for
both KCNMA1 and KCNMB2. A summary of RT-PCR results on tubules from
six KCNMB2⫹/⫹ mice is shown in Table 1. RT, reverse transcriptase.
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KCNMA1 (Mm00516078_m1), KCNMB1 (Mm00466621_m1),
KCNMB2 (Mm00511481_m1), KCNMB3 (Mm01292437_m1),
KCNMB4 (Mm00465684_m1), renin (Mm02342887_mH), hypoxanthine-guanine phosphoribosyltransferase (Mm01545399_m1), and
␤-actin (Mm00607939_s1). Relative mRNA expression of KCNMA1,
KCNMB1, KCNMB2, KCNMB3, KCNMB4, and renin was calculated using the 2⫺⌬Ct method, where Ct is threshold cycle (43). The
average Ct value of hypoxanthine-guanine phosphoribosyltransferase
and ␤-actin was used as the reference Ct value.
Enzymatic digestion and sorting of renal tubules and end-point
RT-PCR. Mice were anesthetized with ketamine-xylazine and perfused with 20 ml PBS through the left ventricle. Kidneys were
harvested, decapsulated, and cut into 8 –10 transverse slices. Kidney
slices were enzymatically digested for 10 min in incubation solution
[NaCl: 140 mM, K2HPO4: 0.4 mM, KH2PO4: 1.6 mM, MgSO4: 1
mM, Na-acetate: 10 mM, ␣-ketogluterate: 1 mM, Ca-gluconate: 1.3
mM, glycine: 5 mM, DNAseI (Roche): 25 mg/l, and collagenase type
II (Pan Biotech, Aidenbach, Germany): 1 mg/ml] for 10 min at 37°C
while being shaken at 850 rpm in a thermomixer (Eppendorf). The
supernatant was aspirated, 1 ml of fresh incubation solution was
added, and incubation was continued in 5-min intervals, with the
removal of the tubule-containing supernatant in between. Tubules
were washed twice in incubation solution with 0.5 mg/ml albumin
(sorting solution) and put on ice. Tubules were sorted to yield
proximal tubule (PT), thick ascending limb (TAL), distal convoluted
tubule (DCT), CNT/CCD, and MCD samples according to their
appearance in phase-contrast microscopy (Fig. 1). PTs were long and
highly convoluted with a smooth appearance. TALs were long, thin,
and straight with a smooth appearance. DCTs and CNT/CCDs were
identified together by the branching points where separate connecting
tubules merge and by a complex appearance, resulting from the
presence of intercalated cells. The transition between the DCT and
CNT/CCD was apparent as an abrupt change in diameter and transition from a slightly convoluted to straight tubule, and DCTs were cut
from CNT/CCDs at this transition. MCDs were identified as long,
straight segments with a complex appearance caused by intercalated
cells, connected to CNT/CCD branching points at the proximal part
and with a progressive narrowing toward the distal part. Twenty-five
PT, 75 TAL, 50 DCT, 50 CNT/CCD, and 50 MCD segments were
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DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
F1038
Table 1. Summary of all end-point RT-PCR experiments on
tubules gathered from six KCNMB2⫹/⫹ mice
Nephron Segment
Proximal tubule
Thick ascending limb
Distal convoluted tubule
Connecting tubule/cortical
collecting duct
Medullary collecting duct
Number of KCNMA1
Bands/Number of
Samples
Number of KCNMB2
Bands/Number of
Samples
6/6
6/6
4/6
5/6
2/6
0/6
3/6
4/6
1/5
0/5
KCNMA1, mice with knockout of the ␣-subunit of the KCa1.1 channel;
KCNMB2, mice with knockout of the ␤2-subunit of the KCa1.1 channel.
Fig. 5. Normokalemic hyperaldosteronism with low renin in KCNMB2⫺/⫺ mice. A: plasma aldosterone concentration in KCNMB2⫹/⫹ (n ⫽ 6) and KCNMB2⫺/⫺
(n ⫽ 6) mice. Data are individual measurements; means are indicated by solid lines. Comparison was made with an unpaired Student’s t-test. B: renal renin
mRNA expression in KCNMB2⫹/⫹ (n ⫽ 6) and KCNMB2⫺/⫺ (n ⫽ 6) mice. Data are individual 2⫺⌬Ct values; means are indicated by solid lines. Comparison
was made with an unpaired Student’s t-test. C and D: plasma K⫹ concentration on the normal diet (C; n ⫽ 6 for KCNMB2⫹/⫹ mice and n ⫽ 8 for KCNMB2⫺/⫺
mice) and high-K⫹ diet (D; n ⫽ 7 for KCNMB2⫹/⫹ mice and n ⫽ 5 for KCNMB2⫺/⫺ mice). Data are individual measurements, means are indicated by solid
lines, and comparisons were made with an unpaired Student’s t-test.
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(Qiagen). cDNA was generated with Superscript III (Invitrogen) and
Superase (Invitrogen). KCa1.1 subunits were undetectable in isolated
tubules using semiquantitative RT-PCR. We therefore performed
end-point RT-PCR of the ␣- and ␤2-subunits of the KCa1.1 channel to
detect low-level mRNA expression of these subunits. End-point RTPCR was performed with HOT FIREPol DNA polymerase (Solis
Biodyne, Tartu, Estonia) and primers against KCNMA1 (forward:
5=-GGAATGCATCTTGGCGTCAC-3= and reverse: 5=-ACAACCACCATCCCCTAAGTC-3=) and KCNMB2 (forward: 5=-TGCAGGACCAACATCCTCTAAG-3= and reverse: 5=-CTTCAGAGCTGTCACAGTTTTCC-3=). PCR products were run on 2% agarose gels for
20 min at 150 V on ice; bands were sequenced to verify amplification
of the correct targets.
Plasma aldosterone. Plasma aldosterone was measured in both
mice fed a normal diet and mice fed a high-Na⫹ diet for 7 days. Mice
were anesthetized by isoflurane inhalation. Once unconscious, mice
were decapitated with sharp scissors. Blood was collected into sample
tubes containing EDTA (Venosafe, Terumo, Belgium) and centrifuged (2,325 g, 3 min at room temperature), and plasma was aspirated
for aldosterone analysis. Aldosterone concentration was analyzed with
an aldosterone ELISA kit (DRG Diagnostics, Marburg, Germany).
Data from this kit were analyzed by four-parameter logistic nonlinear
regression using the online resource MyAssays (www.myassays.
com).
Metabolic cages. K⫹ handling was studied in metabolic cages.
Mice were given food and water ad libitum and kept on the normal
diet for 3 consecutive days followed by 4 days on the high-K⫹/lowNa⫹ diet. Body weight was monitored daily. A 20% decrease was set
as the criterion for the termination of experiments, but no experiments
had to be terminated. Food intake, water intake, urine output, and
fecal output were measured daily.
Plasma K⫹. Mice were anesthetized with ketamine-xylazine, and
blood was sampled by retroorbital puncture using a heparinized
capillary tube (Radiometer, Ballerup, Denmark). Blood was analyzed
immediately in an ABL80 blood gas and osmolyte analyzer (Radiometer, Ballerup, Denmark).
Gavage and K⫹ excretion. Mice were given gastric gavage with a
2% K⫹ (512 mM)-2% sucrose solution to mimic K⫹ ingestion in a
meal (45). K⫹ excretion experiments were performed in both conscious and anesthetized mice with bladder catheters implanted. Experiments in conscious mice are more likely to reflect normal physiology, whereas experiments in anesthetized mice provided higher time
resolution of the urinary K⫹ excretion. For experiments in conscious
mice, animals were given 20 ␮l gavage solution per gram body
weight, equal to ⬃20% of daily K⫹ intake in metabolic cages. Urine
was collected upon micturition, and time was noted. Anesthetized
mice apparently did not tolerate gavage with 20 ␮l/g body wt;
therefore, the gavage volume was reduced to 15 ␮l/g body wt in these
experiments. Anesthesia was induced by and intraperitoneal injection
of ketamine (100 mg/kg body wt) and xylazine (10 mg/kg body wt)
and maintained by intraperitoneal injections of 25% of the initial dose
of ketamine-xylazine every 30 min. Mice were placed on a 37°C
heating plate and had bladder catheters implanted. Urine samples were
collected in 15-min intervals for 3 h. In these experiments, the bladder
was catheterized, and urine samples were collected in 15-min intervals
for 3 h.
Urine analysis. Urinary K⫹ concentration was measured by flame
photometry (Sherwood model 420, Sherwood Scientific, Cambridge,
UK), and total K⫹ excretion was calculated from urinary volume and
K⫹ concentration. To allow comparisons between animals of differing
body weights, K⫹ excretion was calculated as a percentage of the
administered K⫹ load. Urinary osmolality was measured by vapor
pressure osmometry (Vapro 5600, Wescor).
MR blockade. Inspra tablets (Pfizer) were dissolved in tap water (10
mg eplerenone/ml) and administrated by gavage. Mice were given 200
␮g/g body wt daily (divided into two doses of 100 ␮g/g body wt at
9:00 and 21:00) for 4 days before experiments and 100 ␮g/g body wt
at 9:00 on the day of the experiments. The efficacy of the MR
blockade was evaluated in Ussing chambers, as previously described
(46), on distal colonic epithelia from KCNMB2⫹/⫺ mice fed either a
normal diet for 4 days or a normal diet for 3 days followed by the
DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
F1039
Table 2. Plasma chemistries of KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice on the normal diet, after 4 days on the high-K⫹ diet,
and after 4 days of eplerenone treatment
Normal Diet
Number of mice/group
K⫹ concentration, mM‡
Na⫹ concentration, nM
Cl⫺ concentration, mM
Ca2⫹ concentration, mM
HCO⫺
3 concentration, mM
Hematocrit
High-K⫹ Diet
Eplerenone
KCNMB2⫹/⫹
KCNMB2⫺/⫺
KCNMB2⫹/⫹
KCNMB2⫺/⫺
KCNMB2⫹/⫹
KCNMB2⫺/⫺
6
4.17 ⫾ 0.32
148 ⫾ 0.33
105 ⫾ 0.57
1.10 ⫾ 0.056
23.9 ⫾ 1.1
45.0 ⫾ 0.37
8
4.20 ⫾ 0.29
146 ⫾ 0.56
104 ⫾ 0.63
1.12 ⫾ 0.29
24.9 ⫾ 1.2
44.8 ⫾ 0.77
6–7
5.14 ⫾ 0.3
149 ⫾ 1.4
109 ⫾ 2.5
1.07 ⫾ 0.040
25.7 ⫾ 1.5
46.4 ⫾ 2.7
4–5
5.8 ⫾ 0.83
146 ⫾ 0.4
108 ⫾ 2.1†
1.01 ⫾ 0.043
27.6 ⫾ 1.2
43.6 ⫾ 1.3
7–8
4.15 ⫾ 0.13
148 ⫾ 0.65
101 ⫾ 0.61†
1.04 ⫾ 0.023
26.1 ⫾ 0.45
43.1 ⫾ 0.77
8
4.61 ⫾ 0.10*
147 ⫾ 0.40
99.1 ⫾ 0.52*†
1.13 ⫾ 0.016*
28.5 ⫾ 0.9*†
46.6 ⫾ 1.9
Data are shown as means ⫾ SE. Comparisons were made with a Student’s t-test. *P ⬍ 0.05 compared with KCNMB2⫹/⫹ mice on the same treatment;
†P ⬍ 0.05 compared with the same genotype on the normal diet. ‡Plasma K⫹ concentration data from Figs. 5, C and D, and 8.
Flow-induced K⫹ secretion. Mice were anesthetized with ketamine-xylazine as described above, placed on a 37°C heating plate,
and had bladder catheters implanted. High urine flow rates were
induced with satavaptan, obtained from Sanofi under MTA to M.
Bleich at Christian-Albrechts-University. Experiments with satavaptan were designed by M. Bleich. Mice were anesthetized with ketamine-xylazine, and the bladder was catheterized. A baseline urine
sample was collected in a 60-min period, after which satavaptan (3
mg/kg body wt) was administered by an intraperitoneal injection of 47
␮l/g body wt of a 100 ␮M solution of satavaptan in 0.9% saline. A
second urine sample was collected for another 60-min period, initiated
30 min after the satavaptan injection. Mice were not given fluids
during the experiments except for the satavaptan injection and injections for the maintenance of anesthesia.
Fig. 6. Data from metabolic cage experiments. A: urine output normalized to body weight (BW) in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice. B: urine osmolality
in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice. C: urinary K⫹ concentration in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice. D: total urinary K⫹ excretion in KCNMB2⫹/⫹
and KCNMB2⫺/⫺ mice. Data are shown as means ⫾ SE (n ⫽ 5 for KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice); comparisons were made with an unpaired Student’s
t-test. *P ⬍ 0.05 compared with KCNMB2⫹/⫹ mice on the same day.
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aldosterone-inducing high-K⫹/low-Na⫹ diet for 24 h. The aldosterone-inducing diet increases distal colonic ENaC activity, measured as
amiloride-sensitive short-circuit current (Fig. 2). Eplerenone treatment abolished this upregulation of ENaC activity, indicating an
efficient MR blockade.
Blood pressure. Mean arterial pressure (MAP) was measured
noninvasively with a volume-pressure recording (CODA, Kent
Scientific, Torrington, CT). Blood pressure was measured on 5
consecutive days on the normal diet (days 1–5) and 5 consecutive
days on the high-Na⫹ diet (days 8 –12). After measurements on day
5, mice were fed the high-Na⫹ diet. For each mouse, MAP was
calculated as the average of measurements on days 3–5 and days
10 –12; days 1 and 2 and days 8 and 9 were intended as conditioning days to minimize stress artifacts.
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DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
after gavage). In all experiuments, P values of ⬍0.05 were taken to
indicate statistical significance. Statistical analysis was performed
using Graphpad Prism software (version 4.02).
RESULTS
Gross morphology of KCNMB2⫺/⫺ mice. No developmental
or morphological effects of KCNMB2 ablation were observed.
Most experiments were performed on 40- to 60-day-old mice,
and, at this age, there were no differences in the average body
weights [average body weight in male mice: 21.84 g in
KCNMB2⫹/⫹ mice (n ⫽ 12) and 20.92 g in KCMMB2⫺/⫺
mice (n ⫽ 5) and average body weight in female mice: 19.35
g in KCNMB2⫹/⫹ mice (n ⫽ 3) and 19.05 g in KCNMB2⫺/⫺
mice (n ⫽ 6)].
Renal ␤2 mRNA expression. We used RT-PCR to study the
renal mRNA expression of KCa1.1 subunits. Using semiquantitative RT-PCR, ␤2 mRNA was detectable in KCNMB2⫹/⫹
mice but absent in KCNMB2⫺/⫺ mice (Fig. 3). Expression of
␣ mRNA was unchanged, and expression of ␤1 mRNA was
lower in KCNMB2⫺/⫺ mice. Both ␤3 and ␤4 mRNA were
detectable in the whole kidney, albeit at too high Ct values
(Ct ⬎ 38) to allow quantitative comparison of mRNA expression between genotypes. Segment-specific mRNA expression
of ␣ and ␤2 was studied in enzymatically digested tubules
sorted to yield preparations of PTs, TALs, DCTs, CNTs/CCDs,
and MCDs. Using semiquantitative RT-PCR, KCa1.1 ␣- and
␤1–␤4 mRNA expression was undetectable in isolated tubules.
In an attempt to detect low expression levels of KCa1.1 sub-
Fig. 7. Normal urinary K⫹ and Na⫹ excretion in KCNMB2⫺/⫺ mice. A and B: cumulative K⫹ (A) and Na⫹ (B) excretion after oral K⫹ loading by gavage in
conscious mice (n ⫽ 6 for KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice). C and D: cumulative K⫹ (C) and Na⫹ (D) excretion after oral K⫹ loading by gavage in
anesthetized mice (n ⫽ 6 for KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice). Data are shown as means ⫾ SE; comparisons were made with two-way ANOVA.
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Western blot analysis of NCC and ␥-ENaC. Kidneys were homogenized in lysis buffer [sucrose: 0.3 M, imidazole: 25 mM, leupeptin:
8.5 mM, Pefabloc (Sigma): 1 mM, and phosSTOP (Roche Diagnostics, Manheim, Germany): 1 tablet/10 ml]. Samples (20 ␮g protein)
were separated by electrophoresis on Criterion TGX precast gels
(Bio-Rad, Copenhagen, Denmark) and transferred to polyvinylidene
difluoride membranes. Membranes were blocked in 1% BSA in PBS
for 2 h and incubated overnight with anti-total NCC (1:10.000),
anti-phospho-Thr58 NCC (1:5.000), anti-␥-ENaC (1:1.000), or panactin (1:3.000) antibodies in BSA. All antibodies were raised in
rabbits. Anti-total NCC and anti-phospho-Thr58 NCC antibodies were
provided by Jan Loffing (University of Zurich, Zurich, Switzerland).
Anti-␥-ENaC antibody was provided by Jeppe Praetorius (Aarhus
University, Aarhus, Denmark). Anti-pan-actin antibody was purchased from Cell Signaling Technologies. The following morning,
membranes were washed in PBS with 0.1% Tween 20 (PBST),
incubated with horseradish peroxidase-conjugated goat anti-rabbit
IgG in BSA (1:2.000, DakoCytomation, Glostrup, Denmark) for 2 h,
washed in PBST, and developed with Clarity Western ECL (BioRad). Membranes were imaged on an ImageQuant LAS 4000 (GE
Healthcare Life Science), and band intensity was analyzed in Image
Studio Lite (Li-Cor Biosciences). Intensities of total NCC, antiphospho-Thr58 NCC, full-length ␥-ENaC, and cleaved ␥-ENaC bands
were normalized to the intensity of pan-actin bands.
Statistical analysis. Normality of data was tested by the Kolmogorov-Smirnov test. Data were analyzed by an unpaired Student’s t-test
(quantitative PCR, plasma aldosterone, osmolyte analysis, metabolic
cages, plasma K⫹, blood pressure, and Western blots), paired Student’s t-test (satavaptan-induced K⫹ excretion), one-way ANOVA
with Tukey’s multiple-comparison test (efficacy of eplerenone treatment), or two-way ANOVA with a Bonferroni posttest (K⫹ excretion
DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
units, end-point RT-PCR was performed for ␣ and ␤2 mRNA.
␣ mRNA was detectable in all segments except MCD segments
(Fig. 4 and Table 1). ␤2 mRNA was detectable in two of six
PT, four of six CNT/CCD, and three of six DCT preparations
(Fig. 4 and Table 1). These results indicate ␤2 mRNA expression primarily in tubular segments where KCa1.1-dependent
K⫹ secretion has previously been described.
Hyperaldosteronism. KCNMB2⫺/⫺ mice had elevated
plasma aldosterone (342.7 ⫾ 39.03 pg/ml in KCNMB2⫹/⫹
mice vs. 596.4 ⫾ 73.26 pg/ml in KCNMB2⫺/⫺ mice; Fig. 5A).
To determine whether hyperaldosteronism in KCNMB2⫺/⫺
mice resulted from activation of the renin-angiotensin-aldosterone system, we measured renal renin mRNA expression,
which correlates well with plasma renin activity (8).
KCNMB2⫺/⫺ mice had decreased renal renin mRNA expression (Fig. 5B). Aldosterone production by adrenal zona glomerulosa cells can be stimulated by both ANG II and increased
extracellular K⫹ concentration (1, 4, 7). We measured plasma
osmolyte concentrations to determine whether hyperaldosteronism resulted in perturbations in K⫹, Na⫹, Cl⫺, Ca2⫹,
⫺/⫺
HCO⫺
3 concentrations as well as hematocrit in KCNMB2
⫹
mice. There were no differences in plasma K concentration or
any other plasma osmolytes between KCNMB2⫹/⫹ and
KCNMB2⫺/⫺ mice fed a normal diet or a high-K⫹ diet (Fig. 5,
C and D, and Table 2).
Polyuria with lower urinary K⫹ concentration. We studied
K⫹ handling, both on a normal diet and a high-K⫹/low-Na⫹
diet, in metabolic cages. When fed a normal diet,
KCNMB2⫺/⫺ mice displayed polyuria and lower urinary osmolality and K⫹ concentration (Fig. 6, A–C). On high-K⫹/lowNa⫹ diet, both genotypes displayed marked polyuria, a wellknown effect of high dietary K⫹ intake (23), but the differences in urine volume, osmolality, and K⫹ concentration
persisted. Total urinary K⫹ excretion was not different between genotypes on either diet (Fig. 6D). In summary,
KCNMB2⫺/⫺ mice were in K⫹ balance but excreted the
ingested K⫹ in larger volumes of dilute urine.
Urinary K⫹ excretion after oral K⫹ loading. K⫹ excretion
after an acute oral K⫹ load was studied after gavage. Mice
were given a K⫹ load equal to 20% or 15% of the daily K⫹
intake in conscious and anesthetized animals, respectively. The
excretion rate of K⫹ was not different between genotypes in
conscious (Fig. 7A) or anesthetized mice (Fig. 7C), consistent
with comparable K⫹ excretion by KCNMB2⫹/⫹ and
KCNMB2⫺/⫺ mice in metabolic cages. The elevated plasma
aldosterone in KCNMB2⫺/⫺ could, in principle, affect distal
tubular Na⫹ handling; however, Na⫹ excretion induced by the
K⫹ gavage was not different between genotypes in conscious
(Fig. 7B) or anesthetized (Fig. 7D) mice.
Eplerenone-induced mild hyperkalemia and reduced K⫹
excretion. To test whether KCNMB2⫺/⫺ mice required elevated plasma aldosterone to achieve K⫹ balance, we treated
mice with the MR antagonist eplerenone for 4 days. Interestingly, eplerenone treatment induced mild hyperkalemia in
KCNMB2⫺/⫺ mice (4.15 ⫾ 0.13 mM in KCNMB2⫹/⫹ mice
vs. 4.60 ⫾ 0.10 mM in KCNMB2⫺/⫺ mice; Fig. 8). Eplerenone
treatment also induced slightly decreased plasma Cl⫺ concentration and slightly elevated plasma Ca2⫹ and HCO⫺
3 concentrations in KCNMB2⫺/⫺ mice (Table 2). In eplerenone-treated
mice, urinary K⫹ excretion after oral K⫹ loading was significantly slower in KCNMB2⫺/⫺ mice (Fig. 9A). Na⫹ excretion
induced by the K⫹ gavage was marginally but not significantly
lower in KCNMB2⫺/⫺ mice (Fig. 9B). The reduced K⫹ excretion capacity of KCNMB2⫺/⫺ mice was also reflected in
Fig. 9. Decreased urinary K⫹ excretion in eplerenone-treated KCNMB2⫺/⫺ mice. A and B: cumulative K⫹ excretion (A) and cumulative Na⫹ excretion (B) after
oral K⫹ loading by gavage in anesthetized, eplerenone-treated mice (n ⫽ 6 for KCNMB2⫹/⫹ mice and n ⫽ 7 for KCNMB2⫺/⫺ mice). Data are shown as means ⫾
SE; comparisons were made with two-way ANOVA. C: plasma K⫹ concentration at the end of the experiments. Data are individual measurements, means are
indicated by solid lines, and comparison was made with an unpaired Student’s t-test.
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Fig. 8. Plasma K⫹ concentration in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice
(n ⫽ 8 for KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice) after 4 days of eplerenone
treatment. Data are individual measurements, means are indicated by solid
lines, and comparison was made with an unpaired Student’s t-test.
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DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
higher plasma K⫹ concentration in KCNMB2⫺/⫺ mice 3 h
after gavage (Fig. 9C). Thus, eplerenone treatment unmasked a
reduced urinary K⫹ excretion in KCNMB2⫺/⫺ mice.
Blood pressure. Both KCNMA1⫺/⫺ and KCNMB1⫺/⫺ mice
are hypertensive, and hypertension in these strains has been
proposed to stem in part from hyperaldosteronism and from
lack of KCa1.1 activity in vascular smooth muscle cells, leading
to increased myogenic tone (19, 40, 42). We measured blood
pressure in both mice fed a normal diet and mice fed a
high-Na⫹ diet to determine whether hyperaldosteronism results in hypertension in KCNMB2⫺/⫺ mice and whether elevated plasma aldosterone results in the development of saltsensitive hypertension in KCNMB2⫺/⫺ mice. No differences
in MAP were observed between the genotypes, irrespective of
the dietary Na⫹ content (Fig. 10).
Plasma aldosterone on the high-Na⫹ diet. Experiments on
eplerenone-treated mice indicated that KCNMB2⫺/⫺ mice required elevated plasma aldosterone to achieve K⫹ homeostasis.
Therefore, we measured plasma aldosterone in mice fed the
high-Na⫹ diet to determine whether KCNMB2⫺/⫺ mice would
fail to reduce plasma aldosterone upon Na⫹ loading. Both
genotypes were able to reduce plasma aldosterone on the
high-Na⫹ diet; however, KCNMB2⫺/⫺ mice still had increased plasma aldosterone compared with KCNMB2⫹/⫹
mice (Fig. 11).
Fig. 11. Plasma aldosterone concentration in KCNMB2⫹/⫹ (n ⫽ 8) and
KCNMB2⫺/⫺ (n ⫽ 8) mice fed a high-Na⫹ diet for 7 days. Data are individual
measurements, means are indicated by solid lines, and comparison was made
with an unpaired Student’s t-test.
Flow-induced K⫹ secretion. KCNMB2⫺/⫺ mice had a disturbed renal K⫹ excretion. To determine whether FIKS was
affected by ␤2 ablation, we induced diuresis with satavaptan in
a protocol similar to that used by Rieg et al. (38). Both
genotypes displayed FIKS in response to satavaptan-induced
diuresis (Fig. 12), indicating that the ␤2-subunit is not a
prerequisite for FIKS.
Western blot analysis of NCC and ␥-ENaC. Several proteins
involved in renal K⫹ excretion, including NCC, ENaC, and
ROMK, are upregulated by aldosterone and high-K⫹ diets (14,
15, 35, 47). KCNMA1⫺/⫺ mice display hyperaldosteronism,
but renal ROMK protein expression is not increased except in
animals fed a high-K⫹ diet (38). We hypothesized that either
NCC, which is activated by phosphorylation (45, 47), or ENaC,
which is activated by cleavage of ␣- and ␥-ENaC (10, 24, 44),
could be regulated as part of the compensatory mechanism in
KCNMB2⫺/⫺ mice. However, Western blots indicated no
regulation of either total or phosphorylated NCC or full-length
or cleaved ␥-ENaC (Fig. 13).
DISCUSSION
In the present study, we describe renal K⫹ handling in a
KCNMB2⫺/⫺ mouse presenting the interesting phenotype of
normokalemia and hyperaldosteronism, which we show was
secondary to a reduced renal K⫹ excretion. KCa1.1-mediated
K⫹ secretion is now viewed as complimentary to ROMKmediated K⫹ secretion in the CNT/CCD (41). KCa1.1-mediated K⫹ secretion is active under conditions of normal dietary
K⫹ intake, can be stimulated by flow, and can be blocked with
KCa1.1-specific inhibitors (3, 51). Physiological function of
KCa1.1 channels requires coexpression of ␣-subunits with ␤- or
␥-subunits. mRNA expression of ␥-subunits is very low in
human kidneys (54), but mRNA and protein expression of all
four ␤-subunits have been consistently found in the whole
kidney and in the tubular epithelium from different species (11,
18, 31). In accordance with these studies, we confirm mRNA
expression of the ␣- and ␤2-subunit in the CNT/CCD, the site
of regulated K⫹ secretion.
Numerous studies have provided strong evidence for the
involvement of KCa1.1 in renal K⫹ excretion, especially during
conditions of high tubular flow (26, 27, 51). Considering these
studies, it is noteworthy that KCNMA1⫺/⫺ mice, which lack
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Fig. 10. Blood pressure in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice. A: blood pressure on individual days. MAP, mean arterial pressure. B and C: average blood
pressure on the normal diet (B) and high-Na⫹ diet (C). Data are averages of days 3–5 for the normal diet and days 10 –12 for the high-Na⫹ diet (n ⫽ 13 for
KCNMB2⫹/⫹ mice and n ⫽ 11 for KCNMB2⫺/⫺ mice), means are indicated by solid lines, and comparison was made with an unpaired Student’s t-test.
DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
F1043
FIKS, are able to maintain K⫹ balance on both normal and
high-K⫹ diets (38). KCNMA1⫺/⫺ mice have hyperaldosteronism, which is exacerbated by a high-K⫹ diet. The hyperaldosteronism in KCNMA1⫺/⫺ mice was previously proposed to
be of primary origin, resulting from lack of KCa1.1 channels in
the zona glomerulosa of the adrenal gland (42). However, zona
glomerulosa-specific KCa1.1 knockout mice have normal
plasma aldosterone levels (personal communication, Heimo
Ehmke, University of Hamburg, Hamburg, Germany), suggesting that KCa1.1 does not regulate aldosterone secretion. Hypokalemia is commonly observed in both patients (39) and mouse
models with primary aldosteronism (2, 21). An early study (40)
suggested that KCNMA1⫺/⫺ mice were hypokalemic; however, a followup study by Rieg et al. (38) determined that
KCNMA1⫺/⫺ mice are in fact normokalemic. The authors
proposed that hyperaldosteronism in KCNMA1⫺/⫺ mice is
secondary to a decreased KCa1.1-mediated K⫹ excretion and
that KCNMA1⫺/⫺ mice achieve K⫹ homeostasis by upregulation of ROMK-mediated K⫹ excretion.
The importance of individual ␤-subunits for renal K⫹ excretion has been studied in KCNMB1⫺/⫺ and KCNMB4⫺/⫺
mice. KCNMB1⫺/⫺ mice are hypertensive and have hyperaldosteronism and hyperkalemia (19), but this phenotype appears
to be strongly dependent on the genetic background of the mice
(40). Based on 24-h urinary excretion measurements,
KCNMB1⫺/⫺ mice were suggested to have reduced renal K⫹
excretion, leading to hyperkalemia-induced elevation of
plasma aldosterone, which, in turn, contributes to the hypertension (19). KCNMB4⫺/⫺ mice have neither hyperkalemia
nor hyperaldosteronism, but hyperkalemia can apparently be
induced by a high-K⫹ diet (9, 22), indicating a role for ␤4 in
the adaptation to a high-K⫹ diet.
Here, we document that KCNMB2⫺/⫺ mice had hyperaldosteronism and normal plasma K⫹ concentration, a phenotype
Fig. 13. Western blots of the Na⫹-Cl⫺ cotransporter (NCC) and ␥-ENaC. A: Western blots of total NCC (tNCC), NCC phosphorylated at Thr58 (pT58NCC), and
␥-ENaC on whole kidneys from KCNMB2⫹/⫹ (n ⫽ 7) and KCNMB2⫺/⫺ (n ⫽ 6) mice. B: quantification of the band intensities in A.
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Fig. 12. Satavaptan-induced K⫹ excretion in KCNMB2⫹/⫹ and KCNMB2⫺/⫺ mice. A: baseline and satavaptan-induced diuresis. B: baseline and satavaptaninduced K⫹ excretion. Data are shown as means ⫾ SE (n ⫽ 8 for KCNMB2⫹/⫹ mice and n ⫽ 6 for KCNMB2⫺/⫺ mice); comparisons were made with a paired
Student’s t-test.
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DECREASED RENAL K⫹ EXCRETION IN KCNMB2⫺/⫺ MICE
not result in a complete lack of distal tubular large-conductance
K⫹ channel activity. Like KCNMA1⫺/⫺ mice, KCNMB2⫺/⫺
mice must use compensatory mechanisms to achieve K⫹ balance. Unlike KCNMA1⫺/⫺ mice, KCNMB2⫺/⫺ mice could
use KCa1.1 channels composed of ␣-subunits and the other
renal ␤-subunits. The compensatory mechanism in KCNMA1⫺/⫺
mice includes ROMK; however, ROMK is only upregulated in
Western blots when KCNMA1⫺/⫺ mice are fed a high-K⫹ diet.
However, immunohistochemistry of ROMK in the CNT indicates ROMK upregulation in KCNMA1⫺/⫺ mice on both
normal and high-K⫹ diets (38). We performed Western blot
analysis of NCC and ␥-ENaC to determine whether these
proteins are upregulated as part of the compensatory mechanism in KCNMB2⫺/⫺ mice; however, this was not the case.
In summary, we describe the ␤2 subunit of the KCa1.1
channel as necessary for the ability of the kidney to excrete
K⫹. KCNMB2⫺/⫺ mice displayed hyperaldosteronism, which
was secondary to a reduced renal K⫹ excretion capacity. These
results provide a new element in the physiology of renal K⫹
handling that highlights the importance of the KCa1.1 channel
in urinary K⫹ secretion.
ACKNOWLEDGMENTS
We thank Nina Himmerkus for teaching us to sort enzymatically digested
tubules and Jan Loffing and Jeppe Praetorius for providing antibodies for
Western blots. Furthermore, we thank Helle Jakobsen and Karen Sørensen for
technical support. Finally, we also thank Henriette L. Christensen and Christian Westberg for troubleshooting on the RT-PCR.
GRANTS
This work was supported by the Lundbeck Foundation and the Danish
Medical Research Council.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: C.K.L., M.V.S., M.B., and J.L. conception and design
of research; C.K.L., I.S.J., and P.I.d.B. performed experiments; C.K.L., I.S.J.,
and J.L. analyzed data; C.K.L., M.V.S., H.A.P., and J.L. interpreted results of
experiments; C.K.L. prepared figures; C.K.L. and J.L. drafted manuscript;
C.K.L., M.V.S., H.A.P., and J.L. edited and revised manuscript; C.K.L., I.S.J.,
M.V.S., P.I.d.B., H.A.P., and J.L. approved final version of manuscript.
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similar to KCNMA1⫺/⫺ mice. Normokalemia in KCNMB2⫺/⫺
mice indicated that the hyperaldosteronism in KCNMB2⫺/⫺
mice was not of primary origin. Renal renin mRNA expression
was decreased in KCNMB2⫺/⫺ mice, indicating that hyperaldosteronism did not result from renin-angiotensin-aldosterone
system activation. These data suggest that the hyperaldosteronism in KCNMB2⫺/⫺ mice was secondary to a decreased
capacity to excrete K⫹.
This hypothesis was tested by measuring urinary K⫹ excretion. Gavage and metabolic cage experiments clearly demonstrated that KCNMB2⫺/⫺ mice were in K⫹ balance, even when
challenged with acute or chronic K⫹ loads. This was similar to
the observation that KCNMA1⫺/⫺ are normokalemic and use
ROMK channels to compensate for a lack of KCa1.1-mediated
urinary K⫹ excretion (38). To test whether hyperaldosteronism
protects KCNMB2⫺/⫺ mice against hyperkalemia, we treated
mice with the MR antagonist eplerenone. Indeed, eplerenone
treatment induced mild hyperkalemia and a reduced K⫹ excretion rate in KCNMB2⫺/⫺ mice, providing strong evidence for
reduced renal K⫹ excretion in KCNMB2⫺/⫺ mice. These data
suggest that KCNMB2⫺/⫺ mice, like KCNMA1⫺/⫺ mice,
achieve K⫹ balance through aldosterone-induced activation of
a K⫹ secretory mechanism.
Elevated plasma aldosterone is a well-known contributor to
the development of hypertension (20). In this study, however,
we demonstrate that the elevated plasma aldosterone in
KCNMB2⫺/⫺ mice does not have any adverse effects on blood
pressure. KCNMB2⫺/⫺ mice were normotensive, and despite
the well-known antinatriuretic effects of aldosterone,
KCNMB2⫺/⫺ mice did not display salt-sensitive hypertension
when fed a high-Na⫹ diet. Both genotypes were able to
suppress plasma aldosterone when fed a high-Na⫹ diet, although KCNMB2⫺/⫺ mice still had higher plasma aldosterone
than KCNMB2⫹/⫹ mice. These data support that the elevated
plasma aldosterone in KCNMB2⫺/⫺ mice is the result of
physiological regulation of aldosterone production in response
to decreased renal K⫹ excretion. KCNMB2⫺/⫺ mice appropriately suppress plasma aldosterone to achieve Na⫹ balance on
the high-Na⫹ diet but still require slightly higher plasma
aldosterone than KNCMB2⫹/⫹ mice to compensate for the
decreased renal K⫹ excretion and achieve K⫹ balance.
It is noteworthy that KCNMB2⫺/⫺ mice present with elevated plasma aldosterone and reduced renal renin expression,
two traits indicative of primary hyperaldosteronism (12). Although hypokalemia is a classical symptom of hyperaldosteronism, some patients do present with normokalemia (5, 17).
Thus, the elevated aldosterone, low renin, and normokalemia
in KCNMB2⫺/⫺ mice mimic the symptoms of primary hyperaldosteronism. Patients with nonsurgically remediable primary
hyperaldosteronism often benefit from treatment with MR
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KCNMB2⫺/⫺ mice. If this is the case, such patients would not
benefit from treatment with MR antagonists.
In contrast to KCNMA1⫺/⫺ mice, KCNMB2⫺/⫺ mice display intact FIKS, indicating that KCNMB2⫺/⫺ ablation does
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