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*
Division of Endocrinology, Georgetown University, Washington,
DC
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and
Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Physiology, University of Maryland School of Medicine,
Baltimore, Maryland.
Correspondence to Dr. James B. Wade, Department of Physiology, University of Maryland, 655 W. Baltimore Street, Baltimore, MD 21201. Phone: 410-706-3190; Fax: 410-706-8341; E-mail: jwade{at}umaryland.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chronic elevation of vasopressin (AVP) levels is known to cause profound structural (11,12) and functional (13) adaptive changes in the TAL that promote renal concentrating ability. Studies using semiquantitative immunoblotting recently demonstrated that chronic infusion of the AVP analog 1-deamino-(8-D-arginine)-vasopressin (dDAVP) or restriction of water intake causes a consistent and marked increase in NKCC2 expression in the renal outer medulla (14). These treatments seem to selectively affect pathways central to the concentrating mechanism, because consistent increases in expression of the TAL proteins NHE3 and Tamm-Horsfall protein were not observed. NKCC2 expression has also been observed to be increased by chronic NaCl loading (15,16). In view of the central role of apical K+ recycling in TAL function, these studies were undertaken to test the hypothesis that ROMK abundance might also be altered in these adapted states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ROMK Expression in COS Cells and Immunoblotting
ROMK1 and ROMK2 cDNA were subcloned into pCDNA 3.0 (Invitrogen, Carlsbad,
CA) for expression in COS cells. COS cells were grown to approximately 70 to
80% confluence [in Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mM
Hepes] for transfection. After cells were washed once with serum-free DMEM,
they were incubated for 5 h with lipofectamine and 1 µg of DNA in OptiMEM
(Life Technologies-BRL, Grand Island, NY). After the initial incubation with
the transfection mixture, cells were supplemented (at a 1:1 ratio) with DMEM
containing 20% fetal bovine serum and 4 mM L-glutamine. Twenty-four h after
transfection, transfection medium was replaced with maintenance medium
supplemented with 2 mM sodium butyrate, to enhance protein expression.
Forty-eight h after transfection, cells were washed once with ice-cold
phosphate-buffered saline, collected in cold lysis buffer (150 mM NaCl, 20 mM
Tris, pH 7.5, 5 mM ethylenediaminetetraacetate), pelleted (1000 x
g for 5 min), and resuspended in lysis buffer (approximately 5 times
the cell pellet volume) containing 1% Triton and a protease inhibitor cocktail
(10 µg/ml antipain, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, and 10 µg/ml pepstatin A). Cells were then passed through a
27-gauge needle, rotated at 4°C for 1 h, and centrifuged at 15,000 x
g for 15 min at 4°C. Triton-solubilized cell lysate (5 to 20
µg) was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and
transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL).
For Western blotting, L567 and LC35 antibodies were used at 1 to 5 µg/ml.
Horseradish peroxidase-conjugated goat anti-rabbit antibody and rabbit
anti-chicken antibody (Jackson Immunoresearch, West Grove, PA) were used at
1:10,000 for detection with the enhanced chemiluminescence system (Amersham),
according to the recommendations of the manufacturer.
PNGase Digestion
Triton-solubilized COS or kidney cell lysates (20 to 30 µg) were
denatured for 10 min at 60°C in 0.5% sodium dodecyl sulfate/1%
ß-mercaptoethanol, cooled, and then incubated for 60 min at 37°C with
1250 U of peptide N-glycosidase (PNGaseF; New England Biolabs, Beverly, MA) in
50 mM sodium phosphate containing 1% Nonidet P-40.
Animals and Study Design
For these experiments, three different groups of animals were studied. In
the first study, 12 male Brattleboro rats (210 to 260 g; Harlan Sprague
Dawley, Indianapolis, IN), under light methoxyflurane (Metofane;
Schering-Plough Animal Health Corp., Union, NJ) anesthesia, were implanted
with osmotic mini-pumps (Alzet model 2001; Alza Corp., Palo Alto, CA) for
administration of 20 ng/h dDAVP (an AVP V2 receptor-selective agonist)
(n = 6) or saline vehicle (n = 6) for 7 d. In the second
study, male Sprague-Dawley rats (195 to 225 g; Taconic Farms, Germantown, NY)
were water-restricted for 7 d by provision of a limited amount of water as
part of a slurry diet (14).
"Restricted" rats (n = 6) were given an amount of water
chosen to match respiratory losses, i.e., 19 ml water/250 g body wt
per d, mixed with 15 g of powdered food. Control rats (n = 6) were
given 46 ml water/250 g body wt per d, mixed with 15 g of powdered food. Rats
in both groups consumed ail of their food daily and maintained their weight
throughout the 7-d period. This treatment resulted in urinary osmolalities of
549 ± 22 mosmol/kg H2O for control rats and 2913 ±
175 mosmol/kg H2O for water-restricted rats, as previously reported
(14). The effects of the
aforementioned treatments on NKCC2 abundance were previously reported
(14). In the third study, 24
male Sprague-Dawley rats (205 to 255 g) were fed either a control diet (2.75
mEq Na+/250 g body wt per d) (n = 6), a low-sodium diet
(0.25 mEq Na+/250 g body wt per d) (n = 6), a neutral
high-sodium diet (8 mEq Na+/250 g body wt per d) (n = 6),
or an alkaline high-sodium diet (8 mEq Na+/250 g body wt per d,
added as NaHCO3) (n = 6) for 7 d. The effects of a NaCl
diet on NKCC2 in these animals were previously reported
(16). In all studies, rats
were euthanized by decapitation, and both kidneys were rapidly removed and
either frozen on dry ice for later processing or immediately dissected and
homogenized in buffered isolation solution, as described below.
Immunolocalization
Kidneys from ketamine/pentobarbital-anesthetized rats were fixed for
immunolocalization by retrograde perfusion through the abdominal aorta, and
antibodies were immunolocalized on frozen sections as described previously
(17). Sections were incubated
overnight at 4°C with primary antibodies diluted to 10 µg/ml. Secondary
antibodies were species-specific donkey anti-chicken and donkey anti-rabbit
antibodies (Jackson Immunoresearch) coupled to Alexa 488 and Alexa 568,
respectively (Molecular Probes, Eugene, OR).
Immunoblotting of Kidney Samples
Preparation of Samples. Immediately after euthanasia (or after
thawing), kidneys were placed in chilled buffered isolation solution
containing: 250 mM sucrose, 10 mM triethanolamine (Calbiochem, La Jolla, CA),
1 µg/ml leupeptin (Bachem, Torrance, CA), and 0.1 mg/ml
phenylmethylsulfonyl fluoride (United States Biochemicals, Toledo, OH),
adjusted to pH 7.6. Whole right kidneys were homogenized using a tissue
homogenizer (Omni 2000; Omni International Inc., Warrenton, VA), fitted with a
10-mm micro-sawtooth generator, in 10 ml of isolation solution on ice. The
left kidneys were dissected into the cortex and inner stripe of the outer
medulla. Each region was separately homogenized using three bursts of 10 s at
approximately 15,000 rpm, in either 10 ml (cortex) or 1 ml (outer medulla) of
isolation buffer, on ice.
Protein concentrations of the homogenates were measured using the Pierce BCA protein assay reagent kit (Pierce, Rockford, IL). All samples were then diluted with isolation solution to a protein concentration of 1 to 3 µg/µl and were solubilized at 60°C for 15 min in Laemmli sample buffer. Samples were stored at -80°C until gel analysis.
Immunoblotting. To ensure equal protein loading of samples from different rats and treatment groups, each sample set was initially tested by electrophoresis. Five micrograms of protein from each sample were loaded into individual lanes, subjected to electrophoresis on 12% polyacrylamide gels (precast; Bio-Rad, Hercules, CA), and then stained with Coomassie blue dye [G-250 (Bio-Rad); 0.04% solution in 3.5% perchloric acid]. Gels were destained with water, and selected bands were scanned (Scan Jet 6100C; Hewlett Packard, Palo Alto, CA) to determine densities (NIH Image software) and the relative amounts of protein loaded in each lane. When necessary, protein concentrations were adjusted on the basis of these measurements.
For immunoblotting, 10 to 30 µg of protein from each sample were loaded into individual lanes of precast minigels of 7, 10, or 12% polyacrylamide (Bio-Rad). The proteins were electrophoretically transferred from the gels to pure nitrocellulose membranes (Bio-Rad). After 30 min of blocking with 5% milk, membranes were probed overnight at 4°C with the desired affinity-purified polyclonal antibody. For probing blots, all antibodies were diluted into a solution containing 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween-20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondary antibodies were goat anti-rabbit IgG conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and rabbit anti-chicken IgG conjugated to horseradish peroxidase (Jackson Immunoresearch), used at a concentration of 0.1 µg/ml. Sites of antibody-antigen reaction were observed using luminol-based enhanced chemiluminescence (LumiGLO; Kirkegaard and Perry Laboratories) before exposure to x-ray film (Fujifilm; Fugi Medical Supplies, Stamford, CT).
Statistical Analyses
The relative intensities of the resulting immunoblot bands were determined
by laser scanning densitometry (Scan Jet 6100C), followed by analysis using
NIH Image software. The statistical significance of the effects of the various
treatments on expression was determined using an unpaired t test of
densitometry values when SD values were equivalent or using Welch's t
test when SD values were significantly different (GraphPad Prism software;
GraphPad, San Diego, CA). P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It should be noted that there was a small difference in molecular mass between recombinant ROMK and native ROMK in the kidney. This size difference was eliminated by PNGase treatment (Figure 1C), suggesting that it arose from differences in glycosylation. The remaining higher-molecular mass bands observed in the kidney sample after PNGase treatment (Figure 1C) probably resulted from limited effectiveness of the PNGase treatment. Figure 1C also demonstrates that the broad ROMK band observed in the kidney sometimes appeared as a doublet, depending on the gel development conditions; this was also observed with recombinant ROMK2 (Figure 1, A and B).
Immunolocalization of ROMK
The antibody LC35 specifically localizes to the TAL and collecting ducts.
Figure 2 demonstrates
co-labeling with this antibody (Figure 2, A
and B) and the antibody to NKCC2
(Figure 2, C and D). There was
dramatic enhancement of TAL labeling in dDAVP-treated animals
(Figure 2B), compared with
control animals (Figure 2A).
Collecting ducts in the outer medulla were also weakly labeled
(Figure 2). Note that, as
described previously for other antibodies to ROMK
(20,21),
there was distinct heterogeneity of labeling, such that some of the
NKCC2-positive TAL cells were not labeled by the ROMK-specific antibody
(Figure 2, arrows).
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Increased Abundance of ROMK with Chronic dDAVP Infusion
To independently assess whether dDAVP increases ROMK levels, we performed
semiquantitative immunoblotting, comparing the abundance of ROMK in
homogenates of the outer medulla from vehicle- and dDAVP-treated Brattleboro
rats. Figure 3A presents an
immunoblot containing samples from rats given a 7-d infusion of either vehicle
(n = 6) or dDAVP (20 ng/h) (n = 6), probed with the LC35
antibody. Each lane was loaded with a sample from a different rat. As observed
for the transfected COS cells (Figure
1), this antibody exclusively detected a 45-kD band in both groups
of animals. Importantly, immunoreactive ROMK levels consistently increased
with dDAVP treatment. After dDAVP infusion, the average density of the ROMK
band increased by 343 ± 34% (P < 0.005), compared with
vehicle-infused control animals (Figure
3B). Samples from the same animals were also probed with the L567
antibody (Figure 4). Two bands
(45 and 75 to 78 kD) were observed in these outer medullary samples. The
average density of the 45-kD band was also strongly increased in dDAVP-infused
rats (to 593 ± 73% of the value for vehicle-infused rats, P
< 0.001). However, the density of the 75- to 78-kD band did not change with
dDAVP infusion (the average band density for dDAVP-infused rats was equal to
119 ± 11% of the vehicle-treated control mean value). Therefore,
increased labeling of the 45-kD band in immunoblots was correlated with the
striking increase in immunolabeling of the TAL we observed in dDAVP-treated
animals (Figure 2). When
animals were exposed to dDAVP for only 1 h, no significant change in abundance
of the 45-kD band or TAL labeling could be detected (data not shown).
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Effect of Water Restriction on ROMK Abundance
To further assess whether ROMK abundance is increased in antidiuretic
states, we also evaluated ROMK levels in normal Sprague-Dawley rats with
restricted water intake. Water restriction causes an increase in endogenous
levels of circulating AVP and a homeostatic decrease in water excretion by the
kidney. Figures 5 and
6 illustrate the effect of
water restriction (19 ml water/250 g body wt per d) for 7 d, relative to the
water-replete control animals (46 ml water/250 g body wt per d).
Immunolocalization with the LC35 antibody in cross-sections through the outer
medulla demonstrated increased labeling with water restriction, similar to
that observed with dDAVP treatment (Figure
2). Figure 5
illustrates the difference in labeling observed in low-magnification views of
longitudinal sections of the outer medulla. Compared with control animals
(Figure 5A), water restriction
in Sprague-Dawley rats resulted in a strong increase in TAL labeling by the
antibody to ROMK (Figure
5B).
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An immunoblot of outer medullary samples from six control rats (water-replete) and six water-restricted rats is presented in Figure 6A. Water restriction significantly increased the average density of the 45-kD band for the water-restricted rats, to 173% of the value for control rats (P < 0.05) (Figure 6B). A qualitatively similar result was observed when a duplicate blot was probed with the L567 antibody; the density of the 75- to 78-kD band was not affected (data not shown), as in the case of dDAVP infusion.
Changes in Dietary Sodium Alter Abundance of ROMK
In previous studies
(15,16),
we demonstrated that the abundance of NKCC2 is increased by dietary sodium
load. Because of the close relationship of NKCC2 and ROMK with NaCl entry
across the apical plasma membrane of TAL cells, we hypothesized that ROMK
abundance might be altered in response to NaCl intake, in parallel with NKCC2
levels. Figure 7 presents
immunoblots of the whole outer medulla from rats with different levels of
sodium intake as either NaCl or NaHCO3
(Figure 7, A, C, E, and G), as
well as a bar graph summary of the average densitometric findings for the
45-kD band in each blot (Figure 7, B, D, F,
and H). Relatively increased levels of dietary sodium, as either
sodium chloride or sodium bicarbonate, resulted in significantly increased
densities of the ROMK band. No difference in band density was observed for the
45-kD band in rats receiving 8 mEq/250 g body wt per d of NaCl versus
8 mEq/250 g body wt per d of NaHCO3
(Figure 7H).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many groups have described antibodies to ROMK (18,20,21). Although three splice isoforms of ROMK, differing at the amino terminus, have been identified (22,23), currently available antibodies are all directed at conserved sites. Only some of the antibodies developed to ROMK, especially antibodies to the carboxyl terminus, have proven suitable for both immunoblotting and immunolocalization. Although those antibodies localized to the TAL, as expected, they recognized multiple bands (18,20,21). The work presented here takes advantage of newly developed antibodies against ROMK that unambiguously detect ROMK in immunoblots and immunolocalization studies in kidney. In immunoblots of cell lines transfected with ROMK, as well as in the renal outer medulla, ROMK consistently migrates at approximately 45 kD, with variation attributable to glycosylation.
Immunolocalization of ROMK
Immunolocalization assays using the new antibody, LC35, demonstrated strong
localization in the TAL and much weaker localization in collecting duct
principal cells. This confirms previous studies that demonstrated localization
of ROMK at these sites
(18,20,21).
In particular, the strong localization of ROMK in the apical membrane of a
subfraction of TAL cells reflects the cellular heterogeneity of this segment
noted in previous studies
(24,25,26,27).
The functional significance of this cell-type variation remains to be
determined.
Regulation of ROMK Abundance by AVP
The medullary TAL is an important site of action of AVP. Acutely, AVP
increases NaCl transport by this region
(28,29,30).
The K+ conductance of the apical membrane has been demonstrated to
be strongly increased by AVP
(5). Patch-clamp studies
indicate that AVP exposure increases the density of K+ channels,
via a protein kinase A (PKA)-mediated pathway, in both the cortical collecting
ducts and TAL
(31,32).
PKA has been demonstrated to be a regulator of ROMK channels expressed in
oocytes (17), and
site-directed mutagenesis of the three putative PKA sites demonstrated that at
least two of the three sites are required for channel activation
(33). Long-term exposure to
AVP has been demonstrated to have much more dramatic effects in enhancing the
lumen-positive transepithelial potential and ion transport rates, compared
with short-term exposure to AVP
(13). This study indicates
that long-term AVP exposure also has a striking effect on ROMK abundance, as
demonstrated by immunolocalization findings and semi-quantitative
immunoblotting results. These observations reveal a physiologically relevant
mechanism of ROMK regulation that had previously escaped detection.
Outer medullary collecting ducts also express ROMK, and changes in ROMK abundance may occur in this region. However, because of the very high expression of ROMK in the TAL, the changes in ROMK abundance detected in immunoblots undoubtedly reflect changes in TAL rather than collecting duct expression. Further work will be required to determine whether the abundance of ROMK in the collecting duct and other distal regions is also altered by AVP. Previous studies also demonstrated strong effects of long-term AVP treatments on the abundance of the TAL cotransporter NKCC2 (14) and aquaporin 2 in the collecting duct (34). Therefore, AVP contributes to the renal concentrating ability by both short-term and long-term actions. This seems to produce a coordinated increase in expression of the transporters needed to maximally concentrate the urine.
Regulation of ROMK Abundance by Na+ Intake
Because previous investigations noted that NKCC2 abundance could be
affected by Na+ intake
(15,16),
we also assessed the effect of Na+ intake on the abundance of ROMK.
There were strong effects of the high-Na+ diet to increase and the
low-Na+ diet to decrease the abundance of ROMK. Volume expansion
results in a marked increase in NaCl delivery to the TAL
(35) and increased
reabsorption by this segment
(36). Chronic elevation of
Na+ intake has been demonstrated to increase the bicarbonate
absorptive capacity of the medullary TAL
(37). The physiologic role of
enhanced reabsorption by the TAL when overall renal NaCl excretion is
increased is very likely related to the need to maintain water balance even
when urinary osmolality is increased with NaCl. Increased NaCl reabsorption
with recycling of K+ via ROMK tends to elevate interstitial NaCl
levels in the medulla and allow excretion of Na+ loads without
impairment of water balance. Further work is needed to confirm these findings,
but these results suggest that ROMK and NKCC2 may be coordinately regulated by
an unknown mechanism, as well as by AVP.
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Acknowledgments |
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