small-conductance k channel activity was disappeared in the tal and ccd apical membranes of ...

31 Pages·2002·3.44 MB·English

by  Ming Lu

ABSENCE OF SMALL-CONDUCTANCE K+ CHANNEL (SK) ACTIVITY IN APICAL
MEMBRANES OF THICK ASCENDING LIMB AND CORTICAL COLLECTING
DUCT IN ROMK (BARTTER’S) KNOCKOUT MICE

Ming LuΦ, Tong WangΦ, Qingshang Yan, Xinbo Yang, Ke Dong, Mark A. Knepper#, WenHui
Wangξ, Gerhard Giebisch, Gary E. Shull* and Steven C. Hebert
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Department of Cellular and Molecular Physiology, Yale University School of Medicine, New de
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Haven, CT 06520-8026 h
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* Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati w
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College of Medicine, Cincinnati, Ohio 45267-0524 .org
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# Laboratory of Kidney and Electrolyte Metabolism, NHLBI, Bethesda, MD ue
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ξ Department of Pharmacology, New York Medical College, Valhalla, NY 10595 Ma
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Φ Contributed equally to this work 19

Running Title: Absence of SK channel in ROMK knockout mice.
Key Words: small-conductance K channel; thick ascending limb; cortical collecting duct;
ROMK knockout; Kir 1.1; Bartter’s syndrome.

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SUMMARY
The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small-conductance K+
channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-
function mutations in the human ROMK gene cause Bartter’s syndrome with renal Na+ wasting,
consistent with the role of this channel in apical K+ recycling in the TAL that is crucial for NaCl
reabsorption. However, the mechanism of renal K+ wasting and hypokalemia that develop in
individuals with ROMK Bartter’s syndrome is not apparent given the proposed loss of the
collecting duct SK channel. To begin investigation into this latter issue, we generated a colony of
ROMK null mice with ~25% survival to adulthood that provides a good model for ROMK D
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Bartter’s syndrome. The remaining 75% of null mice die in less than 14 days after birth. The ad
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surviving ROMK null mice have normal gross renal morphology with no evidence of h
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hydronephrosis while non-surviving null mice consistently exhibit marked hydronephrosis. w
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ROMK protein expression, assessed using a rabbit anti-rat ROMK polyclonal antibody, was .o
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absent in TAL and CCD from null mice but exhibited normal abundance and localization in u
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wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit M
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consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was 01
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assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/-),
and null ROMK(-/-) mice. In 313 patches with successful seals from the three ROMK genotypes,
SK channel activity in ROMK (+/+ and +/-) exhibited normal single channel kinetics. The
expression frequencies were: 67% (TAL) and 58% (CCD) in ROMK(+/+); about half that of the
wild-type in ROMK(+/-), being 38% (TAL) and 25% (CCD); absent in both TAL or CCD in
ROMK(-/-) between 2-5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK
channel activity in ROMK null mice demonstrates that ROMK is essential for functional
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expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the
normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative
mechanisms for K+ absorption/secretion in the nephron.

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INTRODUCTION
The kidney small-conductance K+ channel (SK) expressed in the apical membranes of
thick ascending limb (TAL) and cortical collecting duct (CCD) cells mediates K+ secretion in the
distal nephron, thereby playing an essential role in K+ balance (1). In the TAL, the SK channel
mediates K+ recycling across the apical membrane that is essential for maintaining the supply of
luminal K+ for Na-K-2Cl cotransport (2-5). In the principal cells of the CCD, the SK channel
provides a major pathway for apical K+ secretion.
ROMK [Kir1.1; gene locus, Kcnj1], cloned from rat kidney outer medulla (6), is a
member of the inward rectifier (Kir) family of K+ channels (7). Several lines of evidence have D
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suggested that ROMK may encode the SK channel. First, there are several NH -terminal ad
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alternative splice variants of rodent (6;8-10) and human (11) ROMK that are differentially h
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expressed along the distal nephron segments where SK channels have been observed (9;10). w
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Second, ROMK protein has been immunolocalized to apical membranes of the nephron .o
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segments expressing the various ROMK transcripts (12-14). Finally, ROMK shares many u
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functional and regulatory properties with native SK channels (15;16). They have similar single M
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channel kinetics including low channel conductance, high open probability, one open time and 01
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one closed time (6;14;16). Moreover, they are similarly regulated by channel phosphorylation
and dephosphorylation processes (17;18), cytosolic pH (19;20), and tyrosine kinases which are
modulated by dietary K+ intake (21-23).
Bartter’s syndrome comprises a set of genetically heterogeneous disorders characterized
by salt wasting and polyuria associated low blood pressure and hypokalemic alkalosis (24-27).
Molecular genetic studies have identified loss-of-function mutations in any one of four genes
encoding transporters mediating salt absorption by the TAL (28-31). Mutations in the apical Na-
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K-2Cl cotransporter [NKCC2; (32)] and basolateral Cl- channel α- [CLCNKB; (33)] or β-
subunits [barttin; (34;35)] result in Bartter’s syndrome by disrupting the pathway for NaCl
transport in the TAL. In addition, null mutations in ROMK also give rise to the Bartter’s
phenotype consistent with a role for this K+ channel in TAL function (36-38). The Bartter’s
mutations in ROMK have been shown to reduce K+ channel expression or function (39-42),
consistent with the necessity of this channel for normal salt reabsorption in the TAL. However,
the characteristic hypokalemia in individuals with ROMK Bartter’s is difficult to explain on the
basis of loss of ROMK function if this channel encodes the SK channel in CCD.
In the present study, we assessed the expression of SK channel by patch clamping in TAL Do
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and CCD from ROMK null mice. Given the low survival and hydronephrosis of the original de
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ROMK null mice [Lorenz et al., JBC 20021] which would have presented difficulties in h
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obtaining tubules for patch clamping, we developed a ROMK null mouse with high survival and w
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normal histology by crossing surviving ROMK null mice with heterozygotes from litters in .org
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which there were surviving null mutants. These mice exhibited characteristics of Bartter’s ue
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including polyuria, Na+ and K+ wasting. Kidney morphology in the ROMK null mice was M
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normal without finding of hydronephrosis, and ROMK protein expression was in TAL and CCD 01
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was absent.
K+ channel activity was absent in apical membranes from either TAL or CCD from
ROMK(-/-) mice while wild-type ROMK(+/+) littermates exhibited normal SK activity. The
percent of successful patches in TAL or CCD showing SK channel activity in heterozygous
ROMK(+/-) mice was ~50% of that in ROMK(+/+) littermates. These results demonstrate that
ROMK encodes the SK channel in apical membranes of both TAL and CCD principal cells.
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EXPERIMENTAL PROCEDURES
Breeding and Genotyping. The generation of the ROMK null mice is described in the companion
paper (Lorenz et al., JBC 20021). Initially, surviving null ROMK(-/-) males were bred with
heterozygous females to enhance survival. ROMK(+/-) heterozygous breeding pairs from these
survivors were intercrossed for several generations to develop a new colony. All mice were
maintained on standard mouse chow and tap water. Pups were genotyped 7 days after birth by
polymerase chain reaction (PCR) using DNA extracted from tail biopsies. The wild-type gene
was amplified using a forward primer (5’-GTGACAGAACAGTGTGCC-3’) corresponding to
codons 149-154 and a reverse primer (5’-CTCCTTCAGGTGTGATGG-3’) corresponding to D
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anticodons 240-234. The mutant gene was amplified using the reverse primer from the ROMK ad
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gene and a primer (5’-CTGACTAGGGGAGGAGTAGAAGG-3’) complementary to sequences h
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in the 5’ untranslated region of the Neo gene. w
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Immunocytochemistry and morphology studies. ROMK(+/+), ROMK(+/-) and ROMK(-/-) mice .o
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were anesthetized by pentobarbital (0.1mg/g body wt) and perfused via the aorta with Hanks’ u
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solution, with drainage from the inferior vena, until the kidneys blanched. The mice were M
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perfusion-fixed with 2% paraformaldehyde/75mM lysine/10mM sodium periodate (PLP). The 01
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kidneys were removed and transferred into 10% sucrose buffer overnight before cutting. Frozen
sections (1µm or 10 µm) were processed for immunofluorescence histochemistry with antibody
labeling (43;44). Sections were incubated for 16-18 h at 4°C with primary antibody [rabbit-anti
rat ROMK produced to amino acids 370-391 in ROMK1; (45)] diluted 1:250 in PBS/0.3% Triton
X-100/0.1% BSA/10% goat serum. After washing 3 times with TBS the sections were incubated
for 1-2 h with goat anti-rabbit Alexa Fluor 488 [IgG (1:200) from Molecular Probes, Eugene,
Oregon USA]. Controls with omission of primary antibody showed no significant fluorescence.
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Metabolic balance. ROMK(+/+) and ROMK(-/-) mice were housed in metabolic cages
obtained from Lab Products Inc, Seaford, Delaware. Two mice from same litter of similar
genotype were housed in a single cage to ensure normal eating and drinking behavior. After 2
days of training in the cage, 24-hour food and water intake and urine output were measured and
recorded. All data represent the average of three 24-hour values. Urinary Na+ and K+
concentrations were measured by flame photometer and daily Na+ (ENa) and K+ (EK) excretion
calculated as mEq/24 hr. Na+, Cl- and K+ concentrations were also measured in plasma from
retro-orbital bleeds by a Corning Blood Gas analyzer (46). Blood gas analysis was performed on
freshly drawn blood and measured by a Corning Blood Gas Analyzer. D
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Patch clamping. Experiments were performed in mice between 2- and 5-weeks after ad
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birth. The left kidney was removed following anesthesia by intraperitoneal injection of h
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pentobarbital sodium (0.1mg/g body wt). 4-5 TAL and CCD tubules were microdissected from w
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each mouse for apical patch clamping as previously described (47). All experiments were carried .o
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out at room temperature (22-24oC). gu
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Bath and tubule-dissection solutions contained (mM): 140 NaCl, 5 KCl, 1.8 MgCl , 1.8 M
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CaCl2 and 10 HEPES (pH 7.4 adjusted with NaOH). For the inside-out patch configuration, the 019
bath solution was the same as the dissection solution except for 0.8 MgCl , 0 CaCl , 1 EGTA.
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0.5mM Mg-ATP (Sigma-Aldrich Co.) was added to the bath to keep channels from running
down. The pipette solution contained (mM): 140 KCl, 1.8 MgCl and 10 HEPES (pH 7.4
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adjusted with KOH).
Single channel activity was recorded in both cell-attached and inside-out configurations
as previously reported (47). For analysis, current recordings were from inside-out patches with a
pipette holding potential (-V) of -40 mV. Channel open time (T ), closed time (T ) and open
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probability (P ) were calculated over 4 sec with a filter frequency of 250 Hz using pCLAMP
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software, version 6.0.5 of Fetchan and pSTAT (Anxon Instrument, Inc.). Channel conductance
was calculated from current-voltage (I-V) curves between -40 mV and –80 mV.
Statistics. Data are presented as means ± SE. Two-way Student's t test was used to compare
control and experimental groups. The difference between the mean values of ROMK(+/+) and
ROMK(-/-) groups was considered significant at P < 0.05.

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RESULTS
ROMK-deficient mice with increased survival without severe hydronephrosis.
Companion studies by Lorenz and coworkers1 showed that the mortality of ROMK-deficient
mice was very high with less than 5% survival to weaning at 21 days. These ROMK(-/-) mice
had renal insufficiency with profound hydronephrosis. We developed a colony with increased
survival by selectively crossing surviving ROMK null mice with heterozygotes from litters in
which there were surviving null mutants. As shown in Fig. 1, 25% of ROMK(-/-) mice survived
to adulthood, the remaining mice died before 14 days. There was no difference in survival rate
between the wild-type ROMK(+/+) and heterozygous ROMK(+/-) mice. D
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Figure 2 shows the gross morphology of kidneys obtained from wild-type ROMK(+/+), ad
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heterozygous (ROMK(+/-), and null (ROMK(-/-) mice that either survived or did non-survive h
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past 14 days. In non-surviving ROMK(-/-) mice, the size of the kidney was about 1/3 of normal w
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(Fig. 2E and F) ; the renal cortex was considerably thinner than in wild-type (Figs. 2A and B) or .o
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heterozygous (Figs, 2C and D) mice, the renal pelvis surrounding the renal papilla and the pelvic u
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fornices at the level of the outer medulla were extensively dilated. These changes indicated that M
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significant hydronephrosis was present in the non-surviving null mice, but this was still 01
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somewhat milder than that seen in the original ROMK-deficient mice (Lorenz et al., JBC 20021).
In contrast, the surviving null mice did not have significant hydronephrosis and the kidneys were
only slightly smaller than those from wild-type (Figs. 2A and B) or heterozygous (Figs, 2C and
D) mice. We cannot exclude the possibly that mild hydronephrosis may be present in a small
number of our ROMK(-/-) mice given the limited number of animals examined in this study.
There were no gross histological difference among the ROMK(+/+), ROMK(+/-) and surviving
ROMK(-/-) mice (Fig. 2).
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Expression of ROMK protein in TAL and CCD is absent in ROMK(-/-) mice. To confirm
the absence of ROMK expression in kidney in our ROMK(-/-) genotype, we examined the
expression and localization of ROMK in wild-type and null mouse kidneys by
immunofluorescence using a polyclonal rabbit anti-rat antibody directed against a COOH-
terminal peptide (45). Figure 3 shows paired phase and immunofluorescence images of ROMK
staining in 1-µm cryosections of kidney cortex from ROMK(+/+) and ROMK(-/-) mice. ROMK
was clearly expressed at apical borders of the TAL and CCD segments in cortical medullary
rays. In contrast, there was no immunostaining of ROMK in null mice in either the TAL or the
collecting duct, confirming the ROMK(-/-) genotype generated as described in the companion Do
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paper (Lorenz et al. JBC 20021). ade
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ROMK-deficient mice have polyuria, increased Na+ and K+ excretion with mild volume h
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depletion but no hypokalemia and a normal acid-base state. Table 1 shows the plasma Na+, Cl- w
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and K+ concentrations measured in adult ROMK(+/+) and ROMK(-/-) mice. Plasma Na+, Cl- and .org
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K+ in ROMK null mice were within the physiological range and similar to wild-type values. gue
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Table 2 shows the acid-base status in ROMK wild-type, heterozygous and knockout mice from
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~2 week old pups and adult mice. The 2-week old animals included both surviving and non- 01
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surviving pups. A slight metabolic acidosis was seen in the ROMK null pups compare to either
ROMK(+/+) of ROMK(+/-) mice. However, no significant abnormality in acid-base status was
observed in adult ROMK null mice.
The results of metabolic studies, shown in Tables 3 and 4, demonstrate that the 24-hour
urine volumes and urinary Na+ and K+ excretion rates were significantly higher in ROMK null
mice compared to wild-type mice. The polyuria in the ROMK(-/-) mice was associated with a
significant reduction in urine osmolality compared to the ROMK(+/+) mice (Table 3). Daily
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