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J Biol Chem, Vol. 273, Issue 50, 33681-33691, December 11, 1998
From To assess whether metabolic acidosis per
se regulates rBSC-1, the rat medullary thick ascending limb
(MTAL) apical
Na+-K+(NH4+)-2Cl Increased urinary NH4+ excretion,
which augments acid excretion, has long been recognized to be
quantitatively the major compensatory response of the kidney against
chronic metabolic acidosis (CMA)1 (1). Stimulation of
proximal tubule ammonium synthesis and secretion was first documented to account for this renal adaptation (2). However, it is established that most of ammonia leaving the
proximal tubule is absorbed by the medullary thick ascending limb
(MTAL), which causes ammonia accumulation in the medullary interstitium
followed by its secretion in adjacent medullary collecting tubules and
thus urinary excretion (2). The role of MTAL ammonia absorption may be
particularly important in states of CMA since the
NH4+ amount absorbed by the loop of
Henle is increased under the latter condition (3, 4). In this regard,
Good (5) has shown that rat MTALs isolated and perfused in
vitro have an increased ability to absorb
NH4+ in response to CMA; this adaptive
response in MTAL would substantially augment
NH4+ excretion during metabolic
acidosis. However, the mechanisms of this MTAL adaptation are unknown.
Absorption of NH4+ by the MTAL occurs by
the activities of several carriers. It is now established that the
luminal step of MTAL NH4+ absorption
involves bumetanide-sensitive
Na+-K+(NH4+)-2Cl The MTAL apical
Na+-K+(NH4+)-2Cl In Vivo CMA--
CMA was induced in male Sprague-Dawley rats in
two ways. In a first series, experimental rats were given 0.28 M NH4Cl in distilled drinking water (8-11
mmol/day), whereas control rats from the same shipment drank distilled
water for 6 days; rats were housed two or three per cage and were
allowed free access to standard rat chow and drinking solution up to
the time of the experiments, at which times the rats were anesthetized
by intraperitoneal injection of sodium pentobarbital. The latter
experimental protocol has been used previously to show the adaptive
increase in MTAL NH4+ absorption in
response to CMA (5).
In a second series, CMA was induced by peritoneal dialysis. Rats were
anesthetized for 3 h by an intramuscular injection of ketamin
chlorhydrate and chlorpromazine (7 and 0.3 mg/100 g in body weight,
respectively) and warmed dialysate was infused into the peritoneal
cavity through an 18-gauge catheter. Control rats were dialyzed against
a solution composed of 110 mM NaCl, 4.5 mM KCl,
35 mM NaHCO3, and 15 g/liter glucose. Rats to
be rendered acidotic were dialyzed against a solution composed of 110 mM NaCl, 4.5 mM KCl, 35 mM
NH4Cl, and 15 g/liter glucose. After 45 min, the dialysate
was removed (95-100% of the initial volume). Rats were then allowed
to have free access to a sodium-, potassium-, and chloride-deficient
diet (in mg/kg of food: 53 Na+, 61 K+, and 43 Cl
In both series, blood was collected by aortic puncture, kidneys were
rapidly removed, and "primary" suspensions of MTALs were prepared
as described below.
Shake Suspension of Rat MTAL Tubules--
The method used to
isolate MTAL fragments has been previously described in detail (25). We
have previously established by light and electron microscopy that this
primary suspension was made almost exclusively of MTALs (
In brief, the MTALs were washed three times by gentle centrifugation
(230 × g for 2 min) at 37 °C in a medium composed
of a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's
modified Eagle's medium (HDMEM) supplemented with 5 mM
heptanoic acid, 5 mM L-leucine, 15 mM Hepes, 0.1 g/liter bovine serum albumin, 400 IU/ml
penicillin, 200 µg/ml streptomycin. The MTALs of the primary
suspension were divided into two equal parts, which gave the control
and acid groups; the HDMEM were supplemented with 10 mM
Tris, 25 mM NaHCO3, pH ~7.35 when gassed with
95% O2, 5% CO2 for the control
group, or 7.5 mM Tris, 15 mM
NaHCO3, pH ~7.10 when gassed with 95% O2,
5% CO2 for the acid group (we have checked that a
difference of 25 mosmol/liter with mannitol at pH 7.35 had no
significant effect on rBSC-1 mRNA abundance by the method described
below (quantitative reverse transcription-PCR; rBSC-1 mRNA was
1.27 ± 0.14 amol/100 ng of RNAtot in control and
1.31 ± 0.11 with additional 25 mM mannitol;
n = 3 for both)). The MTALs of each group were
suspended in the HDMEM at the appropriate pH in 125-ml flasks placed in
a rotary (100 rpm) shaking water bath at 37 °C (hereafter referred
to as shake HDMEM suspension); the shake HDMEM suspensions were gassed
with a humidified 95% O2, 5% CO2 gas mixture
that was filtered through 0.45-µm filter units (NalgeneTM; Nalge Co., Rochester, NY), and were so
maintained for up to 16 h in the dark to protect light-sensitive
components. At the end of the incubation in the HDMEM, MTALs of each
group were washed three times by gentle centrifugation in one of the
media listed in Table I at pH 7.4, and maintained on ice until use.
Samples destined for measurement of intracellular pH (pHi) were loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
acetoxymethyl ester (BCECF-AM), and pHi was estimated at
37 °C as described previously in detail (25).
Preparation of Membrane Vesicles--
A membrane fraction
enriched in apical membrane vesicles was prepared from the MTAL shake
HDMEM suspension by a MgCl2-EGTA aggregation plus
differential centrifugation method, as described previously (24), and
was stored at
Na+-K+-2Cl
For immunoblotting studies, the pooled membrane pellets P2 of each
group were solubilized at ambient temperature for 20 min in Laemmli
medium containing (final concentrations) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol or 5%
2-mercaptoethanol (which made no difference in the results), and 10%
glycerol; the solubilized membranes were immediately used or stored at
We have also prepared crude membranes from primary and shake
suspensions for immunoblotting studies in the following way. MTALs from
control and acid suspensions were homogenized in a medium composed of
125 mM sucrose, 12 mM Trizma (Tris base), pH 7.4, 0.1 mM AEBSF, and 5 µg/ml leupeptin. These
homogenates were centrifuged at 1000 × g for 5 min in
a Sigma 2K15 centrifuge with a 12145 rotor, and the supernatants were
further centrifuged at 210,000 × g for 60 min in a
Beckman L-70 Ultracentrifuge with a 70.1 TI rotor. The pelleted
membranes were solubilized and stored as described above.
Protein and Enzyme Determinations--
Protein amounts were
determined by the method of Bradford (26); alkaline phosphatase
activity was determined from the amount of p-nitrophenol
produced after sample addition from p-nitrophenyl phosphate
used as a substrate (Sigma Diagnostics, Technical Bulletin 104).
Na+/K+-ATPase activity was measured at 37 °C
by a radioisotopic method using [ Electrophoresis and Immunoblotting of Membrane
Proteins--
SDS-PAGE was performed with solubilized membranes
(10-50 µg of protein) and prestained molecular weight markers
(Sigma), on 6-8.5% polyacrylamide minigels (Mini PROTEAN II,
Bio-Rad). Protein was subsequently transferred from the gels
electrophoretically to nitrocellulose membranes (Mini Trans Blot
Module, Bio-Rad). Equal loading and transfer efficiency were
systematically checked by Ponceau Red staining of the nitrocellulose
membranes. Membranes were blocked for 1 h at 37 °C in 5% non
fat milk powder/TBS-T (150 mM NaCl, 10 mM
Tris-HCl, pH 8.5, and 0.1% Tween 20) and exposed overnight at 4 °C
to an anti-rBSC-1 polyclonal antibody (rabbit antibody directed against
a C-terminal rBSC-1 fusion protein; Ref. 18) and to an anti- RNA Extraction, Reverse Transcription, and Polymerase Chain
Reaction--
MTAL total RNA (RNAtot) was extracted from
aliquots of primary and shake suspensions by the method of Chomczynski
and Sacchi (28). To obtain a competitor RNA (cRNA) that differed from
the wild rBSC-1 mRNA by a 116-base deletion of the latter, the 5' end of the rBSC-1 cDNA (bp 1-575) was inserted between the
ApaI and SalI restriction enzymes sites of the
Multicloning Site of pBluescript® SK+ (Stratagene Inc., La
Jolla, CA) and subcloned with use of DM1TM competent cells
(Life Technologies Inc.) to select an unmethylated plasmid, hereafter
referred to as the 5' rBSC-1 cDNA plasmid. A 116-bp deletion (bp
393-509 of the rBSC-1 sequence) of the 5' rBSC-1 cDNA plasmid was
obtained by digestion with StuI and MscI restriction enzymes, followed by self-ligation of the cut plasmid. The
deleted 5' rBSC-1 cDNA plasmid was subcloned, linearized with KpnI restriction enzyme, and 3' blunt-ended with the Klenow
fragment of E. coli DNA polymerase I. In vitro
transcription was then performed with use of T3 RNA polymerase
(mCAPTM RNA capping kit; Stratagene) and
[32P]UTP. The amount of transcribed cRNA was determined
by the measure of its optical density at 260 nm corrected for the ratio
of trichloroacetic acid-precipitated cRNA to total cRNA determined by
liquid scintillation spectroscopy.
The primers (Genset) for cDNA synthesis and PCR amplification were
chosen from the published rBSC-1 sequence (GenBank accession no.
U10097) with the help of Oligo® 4.04 primer analysis
software (National Biosciences Inc., Plymouth, MN). The sequences of
the primers were: 5'-CCA AAA CCA AGT GCT CGG TAT T-3' (sense; bases
107-128) and 5'-GGT GTT GCG GTA GTA CTC AAT C-3' (antisense; bases
536-557). These primers yielded, as expected, only one PCR product of
451 bp from MTAL RNAtot or the undeleted 5' rBSC-1 cDNA
plasmid, and of 335 bp from the cRNA or the deleted 5' rBSC-1 cDNA
plasmid, respectively; after 30 PCR cycles, PCR products resulting from
nonspecific hybridization were never observed. The identities of the
PCR products were confirmed after digestion by the restriction enzyme
HincII by PAGE, followed by Southern blot analysis with a
rBSC-1-specific 32P-labeled oligonucleotide probe; the
sequence of this probe was 5'-CAC CAA CCA CTG TAA AAT AGG T-3' (bases
172-193).
cDNAs were synthesized from MTAL RNAtot and cRNA by
reverse transcription (RT) at 37 °C for 60 min with 200 units of
Moloney murine leukemia virus reverse transcriptase (Life Technologies Inc.), 20 pmol of antisense primer, 4 µg of yeast transfer RNA, 2.5 mM of each deoxyribonucleotide (dNTP), 10 mM
dithiothreitol, 2 units of ribonuclease inhibitor, and 4.4 µl of 5×
RT buffer in a final volume of 22 µl. Reverse transcriptase was then
inactivated at 95 °C for 5 min. Each reaction was performed in
parallel with an otherwise identical one that contained no reverse transcriptase.
For PCR, 10 µl of the cDNA solution were supplemented with 5 µl
of 10× PCR buffer, 5.6 mM MgCl2, 40 pmol of
sense and antisense primers, 0.5 mM of each dNTP, and 1.25 units of Taq DNA polymerase (Life Technologies Inc.) in a
final volume of 50 µl. Samples were overlaid with mineral oil and
denatured at 95 °C for 4 min, which was followed by cycles
consisting of denaturation at 95 °C (1 min), annealing at 58 °C
(1 min), and extension at 72 °C (1.5 min); PCR was completed by a
final extension step at 72 °C for 10 min. Quantitative PCR was
performed using 24 cycles of amplification of cDNAs simultaneously
obtained from a fixed amount of MTAL RNAtot (10-150 ng, as
appropriate) and 0.27-2 amol of cRNA. We have established these PCR
conditions in preliminary experiments to prevent heteroduplexes of PCR
amplicons from occurring (heteroduplexes were apparent with Materials--
Carrier-free 22NaCl,
[ Statistics--
Results are expressed as means ± S.E.
Statistical significance between experimental groups was assessed by
Student's paired or unpaired t test or by one-way ANOVA
completed by a t test using the within-groups residual
variance of ANOVA, as appropriate.
In Vitro Studies in Shake Suspensions
Transport Studies in Intact MTAL Tubules--
We have previously
established that MTALs from shake suspensions were viable and retained,
as assessed by electron microscopy examination, their normal tubular
structure with only slight ultrastructural changes compared with MTALs
from the primary suspension (24). We have checked, by the method
described previously (24), that there was no difference in the electron
microscopy appearance3
between MTALs from the control and acid groups.
Na+-K+(NH4+)-2Cl
To assess the cellular mechanisms responsible for the results described
above, MTALs were incubated in control or acid media containing 20 µM actinomycin D or 20 µM cycloheximide to
block gene transcription or protein synthesis, respectively. As shown in Fig. 2C, actinomycin D or cycloheximide alone tended to
reduce Na+-K+(NH4+)-2Cl
The intrinsic cell buffering power (
Thus, it appeared that in vitro acid pretreatment stimulated
the
Na+-K+(NH4+)-2Cl Transport Studies in Membrane Vesicles--
To assess whether acid
incubation directly stimulates Na+
K+(NH4+)-2Cl
Specific activities, enrichment factors, and yields of marker enzymes
in nine control and acid membrane preparations are summarized in Table
III. We have previously established
alkaline phosphatase as a marker enzyme for the MTAL apical membrane
(12, 24). The membrane preparations from control and acid MTALs were
both enriched approximately 7-fold in apical membrane, the yield of which was about 20% in both groups, and were identically contaminated by basolateral membrane as seen in similar enrichment factors and
yields in Na+/K+-ATPase. It must be emphasized
that alkaline phosphatase and Na+/K+-ATPase
activities in the homogenates were not modified by the acid
pretreatment (Table III), which indicates that acid incubation did not
generally stimulate all transport and enzyme activities of MTAL
cells.
Na+-K+-2Cl
These results indicate that acid pretreatment of MTALs directly
stimulated
Na+-K+(NH4+)-2Cl Effect of Acid Incubation on rBSC-1 Protein Abundance
rBSC-1 protein abundance was determined in MTAL apical
membrane-enriched and crude membranes from the shake suspensions by Western blot analysis using an affinity-purified polyclonal antibody directed against a C-terminal rBSC-1 fusion protein (18); Effect of Acid Incubation on rBSC-1 mRNA Abundance
rBSC-1 mRNA abundance was determined by quantitative RT-PCR
using an in vitro transcribed cRNA that differed from the
wild mRNA by a 116-bp deletion. A typical quantitative RT-PCR is
illustrated in Fig. 7. As shown in Fig.
8, rBSC-1 mRNA was increased by acid incubation in each of seven preparations, with mean values of 0.245 ± 0.017 amol/100 ng of RNAtot in control group
and 0.384 ± 0.025 in acid group (p < 0.0001),
which represented a 58 ± 7% increase.
Stimulation by in Vivo and in Vitro
Metabolic Acidosis of Expression of rBSC-1, the
Na+-K+(NH4+)-2Cl
Cotransporter of the Rat Medullary Thick Ascending Limb*
,,,
, and**
INSERM Unité 356, Physiologie et
Endocrinologie Cellulaire et Moléculaire Rénale,
Université Pierre et Marie Curie, 75006 Paris, France and the
§ Division of Nephrology, Vanderbilt University, Nashville,
Tennessee 37232-2372
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and
membrane vesicles by intracellular pH and 22Na+
uptake measurements, respectively; rBSC-1 protein was quantified by
immunoblotting analysis and mRNA by quantitative reverse
transcription-polymerase chain reaction. As compared with incubation at
pH ~7.35, acid incubation (pH ~7.10) up-regulated by 35-100%
rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1
protein and mRNA abundance. In contrast, acid incubation did not
alter alkaline phosphatase and Na+/K+-ATPase
enzyme activities or 
-actin protein abundance. After 3 h of
in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA
abundance increased in freshly harvested MTALs, which was accompanied
after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in
vitro CMA stimulate rBSC-1 expression, which would contribute to
the adaptive increase in MTAL absorption and urinary excretion of
NH4+ in
response to CMA.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cotransport (6-10) and barium- and verapamil-sensitive
K+/NH4+(H+)
antiport (11, 12); an amiloride-sensitive
NH4+ channel has also been described in
the MTAL apical membrane (11, 12) but its contribution to
NH4+ absorption is probably minor since
this channel is also permeable to Na+ and
K+.
cotransporter was recently cloned from rat (13), mouse (14, 15), rabbit
(16), and human (17) kidneys, and was named BSC-1 (bumetanide-sensitive
cotransporter) or NKCC-2
(Na+-K+-Cl cotransporter).
Antibodies to BSC-1 localize the transporter protein at the apical
membrane of the cortical and medullary TAL, as well as the macula densa
(18, 19). The promoter of the murine BSC-1 gene contains several
consensus sites for various transcription factors, which suggests that
transcription of BSC-1 is responsive to physiological stimuli (20, 21).
Indeed, BSC-1 was recently shown to be up-regulated by chronic saline
loading (19) and down-regulated by potassium depletion (22), but not affected by water deprivation and arginine vasopressin administration (19, 23). That BSC-1 mediates the majority of MTAL
NH4+ absorption in addition to NaCl
absorption suggests that BSC-1 could be regulated by acid-base status;
however, this hypothesis has not yet been tested. These considerations
prompted us to design the present study to assess whether acidosis
per se, simulated in vitro with use of an acid
medium, affects rBSC-1, the rat MTAL cotransporter (13). To this end we
have measured the effects of a 16-h incubation of the MTAL "shake"
suspension (24) in an acid-defined medium on rBSC-1 transport activity,
protein, and mRNA. We have also determined the abundance in MTAL of
rBSC-1 protein and mRNA after in vivo CMA. The results
show for the first time that rBSC-1 expression is up-regulated by both
in vivo CMA and in vitro acid incubation.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
) and to a drinking solution containing 60 mM NaCl, 60 mM KCl, 27 mM
KHCO3, and 2.5 g/liter glucose for control rats, or 60 mM NaCl, 87 mM KCl, 50 mM
NH4Cl (1.5-2 mmol/day), and 2.5 g/liter glucose for
acidotic rats (all rats were given the above control diet and drinking
water for 5-7 days before peritoneal dialysis). Rats were
re-anesthetized 1 or 2 days after the beginning of peritoneal dialysis
for the experiments.
95%),
occasional thin limbs, and rare outer medullary collecting tubules,
with no isolated cells or proximal tubules (24). All of the following
steps of the preparation of the suspensions were performed under
sterile conditions with use of filter-sterilized media, as described
previously in detail (24).
80 °C and used within 2 weeks.
cotransport activity
was assessed at ambient temperature (22-25 °C), after the membrane
vesicles were loaded with solution C in Table I, by a filtration
technique using 0.45-µm cellulose acetate-nitrate filters (Millipore,
HAWP), as described previously in detail (12, 24).
80 °C.
-32P]ATP, as
described previously (12).
-actin
mouse monoclonal antibody (clone AC-15, Sigma-Aldrich Fine Chemicals)
diluted 1:500 and 1:100,000, respectively, in 5% nonfat milk
powder/TBS-T. Anti-rBSC-1 polyclonal antibody was affinity-purified as
described (27). The membranes were then exposed to the second
antibodies (peroxidase-linked anti-rabbit Ig and anti-mouse Ig;
Bio-Rad) for 1.5 h at ambient temperature. After washing in TBS-T,
antigen-antibody complexes were detected by autoradiography using
enhanced chemiluminescence (ECL Western blotting Analysis System,
Amersham Life Science). The autoradiography was digitized with use of a
laser scanner (ScanJet IIcx, Hewlett-Packard), and quantification of
each band was performed by densitometry with use of the public domain
NIH Image 1.52 software2; for
one experiment, rBSC-1 and -actin proteins of control and acid
groups were quantified in duplicate and the densitometry values were
normalized so that the mean value of the control group was 100 arbitrary units to facilitate comparisons. For immunoabsorption studies, the affinity-purified anti-rBSC-1 antibody was incubated with
the fusion protein for 3 h at room temperature. An example of
rBSC-1 and -actin protein detection is illustrated in Fig. 1. In
some membrane preparations, a high molecular weight band was observed
at the top of the gel in addition to the expected rBSC-1 band of ~150
kDa (18); the high molecular weight band was likely composed of rBSC-1
protein aggregates, since it was not always observed and reactivity was
abolished by pre-adsorption of the antibody with rBSC-1 fusion protein.
Indeed, control experiments with affinity-purified anti-rBSC-1 antibody
pre-adsorbed with fusion protein gave no labeling for both the
~150-kDa and high molecular weight bands (Fig. 1); the rBSC-1 bands
were also not observed with the pre-immune rabbit serum (not
shown). -Actin protein was visible as a ~42-kDa band (Fig.
1).
View larger version (49K):
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Fig. 1.
Immunoblots of rBSC-1 and -actin proteins
in two membrane preparations from MTAL shake suspension.
Lane 1, in this example of apical
membrane-enriched preparation, rBSC-1 protein appeared as one strong
band centered at ~150 kDa and a faint band that probably represented
rBSC-1 protein aggregates at the top of the gel. Lane 2,
rBSC-1 bands were not seen after preabsorption of the anti-rBSC-1
antibody by the fusion protein. -Actin protein appeared as one
42-kDa band in both preparations. Each lane of these 8.5%
polyacrylamide gels was loaded with ~15 µg of protein.
28 cycles
under otherwise identical conditions). The PCR amplicons were resolved
by PAGE and stained with ethidium bromide for 45 min at ambient
temperature. A photograph of the gel was digitized using the laser
scanner, and quantification of the bands was performed by densitometry
with use of the NIH Image 1.52 software. To correct for differences in
molecular weight, the densitometry values of the competitive DNA bands
were multiplied by the 451/335 bp ratio. We have checked that the
amplification efficiencies of the wild and competitive cDNA were
identical with up to 27 PCR cycles (0.35 ± 0.04 versus
0.38 ± 0.05 per cycle, n = 6 for both) and thus
that no further correction was needed, and that the amounts of
amplicons obtained after 24 PCR cycles were well within the exponential
phase of amplification. The rBSC-1 mRNA abundance was calculated
from the linear log-log scale plot of the ratio of the fluorescence
intensities of cRNA to MTAL RNAtot PCR products against the
known amount of cRNA added in each reaction tube; r values
for these linear plots were all >0.99 (see Fig. 7 below). Results are
expressed in attomoles of rBSC-1 mRNA/100 ng of
RNAtot.
-32P]ATP, and [32P]UTP were obtained
from Amersham, Buckinghamshire, UK; guanidium thiocyanate, phenol,
Taq DNA polymerase, Moloney murine leukemia virus reverse
transcriptase, and restriction enzymes were from Life Technologies,
Inc.; dNTP and yeast transfer RNA were from Stratagene. Collagenase CH
grade II was obtained from Boehringer Mannheim S.A., Maylan, France; BCECF-AM was from Molecular Probes, Eugene, OR;
D()-mannitol was from Merck, Darmstadt, Germany. AEBSF,
leupeptin, amiloride, bumetanide, EGTA, Tris-ATP, and all other
chemicals were obtained from Sigma-chimie S.A.R.L.,
LaVerpillière, France.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cotransport activity was assessed by determining the
bumetanide-sensitive component of the cell acidification caused by
abrupt exposure to 4 mM NH4Cl, as described
previously (11, 29). MTALs from control and acid groups were removed from the HDMEM, washed three times, and suspended in
CO2/HCO3-free solution A
in Table I, loaded with BCECF, and
maintained on ice until the pHi measurements. After warming and washing by gentle centrifugation, samples of MTALs were diluted in the
fluorometer cuvette in 2 ml of solution B in Table I and pHi
was monitored at 37 °C. Before NH4Cl addition, resting pHi was slightly lower in acid-preincubated than in control MTALs, which might have been explained by an effect of prolonged acid
incubation on cell metabolism leading to an increased metabolic production of metabolic acids. In the presence of 10 mM
barium + 1 µM amiloride to block
NH4+ carriers other than
Na+-K+(NH4+)-2Cl
cotransport such as barium-sensitive
K+(NH4+)-Cl
cotransport and
K+/NH4+(H+)
antiport and amiloride-sensitive NH4+
conductance (10, 11, 29), addition of 4 mM
NH4Cl caused a rapid cell alkalinization due to
NH3 entry, which ended when intracellular and extracellular
NH3 concentrations were equal (Fig.
2A). This was followed by cell
acidification caused by NH4+ entry and
subsequent dissociation within the cells coupled to NH3
exit (Fig. 2A); we have demonstrated previously that the
initial rate of NH4+-induced cell
acidification (dpHi/dt, calculated as described previously; Ref. 29) is not significantly affected by changes in the
activities of pHi regulatory mechanisms such as Na+/H+ antiport (11, 29). In the presence of 10 mM barium + 1 µM amiloride, acid incubation
increased the NH4+-induced
dpHi/dt from 0.34 ± 0.05 pH unit/min in
control to 0.70 ± 0.05 (p < 0.0003; Fig. 2,
A and B). In contrast, in the presence of 10 mM barium + 1 µM amiloride + 0.1 mM bumetanide to also block
Na+-K+(NH4+)-2Cl
cotransport, the cell acidification was abolished and replaced by a
simple return toward the basal pHi value at 0.14 ± 0.02 pH unit/min in control group and 0.10 ± 0.01 in acid group (p < 0.05; Fig. 2B). Thus, when
Na+-K+(NH4+)-2Cl
cotransport was blocked, the rate of recovery toward basal pHi was, on the contrary, slightly decreased by acid incubation. The latter
observation could have been explained by stimulation of the MTAL apical
Na+/H+ antiporter NHE-3 since MTAL NHE-3 was
demonstrated to be up-regulated by metabolic acidosis (30); this
phenomenon was beyond the scope of the present study and was not
further investigated.
Composition of experimental solutions
View larger version (28K):
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Fig. 2.
Na+-K+(NH4+)-2Cl
cotransport activity in intact cells. MTALs from control or acid
shake suspensions were incubated in CO2-free medium, pH
7.4, and pHi was monitored. Panel A, addition at
time zero of 4 mM NH4Cl caused an immediate
cell alkalinization (NH3 entry and equilibration) followed
by cell acidification due to NH4+ entry
coupled to NH3 exit; in the presence of 1 µM
amiloride + 10 mM barium,
NH4+ entry occurs via
Na+-K+(NH4+)-2Cl
cotransport, which was stimulated by acid incubation. Panel
B, in the presence of 1 µM amiloride + 10 mM barium, acid incubation of MTALs enhanced the
NH4+-induced initial rate of cell
acidification (dpHi/dt) from 0.34 ± 0.05 pH
unit/min in control to 0.70 ± 0.05 (p < 0.0003), but not in the additional presence of 0.1 mM
bumetanide to block the cotransporter. Panel C, 20 µM actinomycin D or cycloheximide prevented acid
incubation from significantly enhancing
NH4+-induced dpHi/dt
in the presence of 1 µM amiloride + 10 mM
barium. Statistical comparisons were made by unpaired Student's
t test (panels A and B) and
ANOVA (panel C).
cotransport activity, but this did not reach statistical significance. Acid incubation did not enhance
Na+-K+(NH4+)-2Cl
cotransport activity in the presence of actinomycin D or cycloheximide (Fig. 2C).
i) was estimated, as
described previously (29), from the magnitude of the intracellular alkalinization following abrupt addition of 12 mM
NH4Cl in the presence of 10 mM barium + 1 mM amiloride + 0.1 mM bumetanide + 2 mM ouabain to block all NH4+
carriers (10, 11); under these experimental conditions, cell alkalinization due to NH3 entry was followed by a plateau
acidification. As shown in Table II,
i was not different in control and acid MTALs;
i values were also not different from those measured
previously in MTALs from primary suspensions (29).
Intrinsic cell buffering power (i)
i was determined from the magnitude of cell alkalinization
caused by abrupt exposure to 12 mM NH4Cl of MTALs
that were incubated in solution B of Table I containing 10 mM barium + 1 mM amiloride + 0.1 mM bumetanide + 2 mM ouabain to block all
NH4+ carriers (these agents were applied to the cells 3 min before the measurements); pHi values are the mean
pHi values (middle of pHi before and after
NH4Cl addition) at which i was determined. NS, not
significant.
cotransport activity in intact MTAL cells. However, cotransport activity in intact cells could have been stimulated directly and/or indirectly, for example through changes in
Na+/K+-ATPase or other carriers activities and
transmembrane ion gradients.
cotransport, transport studies were conducted in membrane vesicles enriched in MTAL apical membrane.
Specific activities, enrichment factors, and yields of marker enzymes
in fractions obtained during the isolation procedure
cotransport activity
in the vesicles was measured as the bumetanide-sensitive 100 mM KCl gradient-stimulated 0.5 mM
22Na+ uptake, as described previously (12, 24).
As shown in Fig. 3A, the mean
value of total 22Na+ uptake during 10 s
was significantly higher in acid than in control vesicles
(p < 0.035), whereas there was no difference in the
presence of bumetanide; the residual bumetanide-insensitive
22Na+ uptake was largely due to
22Na+ external binding to membranes, as
previously documented (12). Equilibrium values of
22Na+ uptake were identical in control and acid
vesicles both in the presence and absence of bumetanide (Fig.
3B), which indicates that the difference in total
22Na+ uptake observed after 10 s was not
due to a difference in 22Na+ binding to the
membranes. Thus, the bumetanide-sensitive component of
22Na+ uptake, which was due to
Na+-K+-2Cl cotransport activity
as previously demonstrated (12), was enhanced by acid incubation in
each of 7 membrane preparations, with mean values of 152 ± 14 pmol of 22Na+/mg of protein/10 s in control
group and 205 ± 21 in acid group (p < 0.0025;
Fig. 4), which represented a 35 ± 8% increase.
View larger version (16K):
[in a new window]
Fig. 3.
Na+-K+(NH4+)-2Cl
cotransport activity in membrane vesicles. Vesicles were loaded
with solution C and diluted in solution D (solutions are described in
Table I) containing 0.5 mM 22Na+
and 100 mM KCl to activate
Na+-K+(NH4+)-2Cl
cotransport. Panel A, after 10 s, acid
incubation enhanced 22Na+ uptake in the absence
(total) but not presence of 1 mM bumetanide. Panel
B, at equilibrium (2 h), there was no difference in
22Na+ uptake between the various experimental
conditions. Each bar represents the mean ± S.E. of
measurements in triplicate in seven (panel A) and four
(panel B) membrane preparations.
View larger version (20K):
[in a new window]
Fig. 4.
The bumetanide-sensitive component of
22Na+ uptake
[Na+-K+(NH4+)-2Cl
cotransport activity] was calculated from data shown in panel A
of Fig. 3. Acid preincubation stimulated
Na+-K+(NH4+)-2Cl
cotransport activity in each of the seven membrane preparations.
Statistical comparison was made by paired Student's t
test.
cotransport activity.
-actin protein abundance was also determined in these membrane preparations. Fig. 5 shows results obtained in
representative preparations of apical membrane-enriched and crude
membranes, and the mean results for the whole study are summarized in
Fig. 6. With respect to the quantitative
effects of acid incubation, there was no clear difference in
acid-induced increases in rBSC-1 protein abundance between apical
membrane-enriched (+~36%; n = 3) and crude membranes (+~53%; n = 4) and thus the data were pooled.
Whereas the intensity of the 42 kDa -actin band was not
significantly affected by in vitro acid incubation (100 ± 4 arbitrary units versus 105 ± 8; n = 12 for both), that of the rBSC-1 protein was enhanced by acid pretreatment in each of seven preparations, with mean values of 100 ± 6 arbitrary units in control group and 146 ± 8 in
acid group (n = 14 for both, p < 0.0001; Fig. 6).
View larger version (54K):
[in a new window]
Fig. 5.
Immunoblots of rBSC-1 protein in
representative apical membrane-enriched (panel 1) and crude
(panel 2) membrane preparations. In these examples,
acid incubation increased rBSC-1 protein abundance by 46%
(panel 1) and 60% (panel
2). Each lane of these polyacrylamide gels was loaded with
13 (panel 1) and 30 µg of protein (panel 2).
Ac., acid preincubation; C, control.
View larger version (19K):
[in a new window]
Fig. 6.
Band densities of immunoblots of rBSC-1 and
-actin proteins in control and acid membranes. Bars
represent means ± S.E. of measurements in duplicate in seven and
six membrane preparations for rBSC-1 and -actin, respectively.
Statistical comparisons were made by unpaired Student's t
test.
View larger version (24K):
[in a new window]
Fig. 7.
Quantification of rBSC-1 mRNA by
quantitative RT-PCR. A digitized reproduction of an entire
ethidium bromide-stained polyacrylamide gel is shown in the upper
panel; RT-PCRs (24 PCR cycles) were done with 125 ng of MTAL
RNAtot in each reaction tube and, from left to right, 2.00, 1.43, 1.02, 0.73, 0.52, 0.37, and 0.27 amol of cRNA; RT ,
reaction done in the absence of reverse transcriptase. The lower
panel shows the log-log plot of the ratio of densitometry values
(335/451) against initial amount (attomoles) of cRNA; in this example,
initial rBSC-1 mRNA amount was 0.43 amol/100 ng of
RNAtot.
View larger version (19K):
[in a new window]
Fig. 8.
Acid incubation-enhanced rBSC-1 mRNA in
seven preparations, as assessed by quantitative RT-PCR.
Statistical comparison was made by paired Student's t
test.
Time Course of the Effect of Acid Incubation on rBSC-1 mRNA Abundance
We have shown previously that MTALs incubated at pH 7.35 for
16 h in shake suspensions exhibited virtually unchanged
Na+-K+(NH4+)-2Cl
cotransport, Na+/H+ exchange, and
Na+/K+-ATPase activities as compared with those
observed in primary suspensions (24). This suggests that the abundance
of the proteins responsible for the latter transport activities remains
relatively stable in MTAL cells of the shake suspensions. In the
present study, we have assessed the evolution in function of time of
rBSC-1 mRNA abundance in shake suspensions as compared with that in
primary suspensions. The results are summarized in Fig.
9, which shows that rBSC-1 mRNA
abundance in MTAL cells in shake suspensions decreased to reach a new
stable value within the first 9 h of incubation. Thus, the present
and previous observations suggest that MTAL cells adapted themselves in
a first period that lasted <9 h to the new in vitro
environment with respect to rBSC-1 mRNA abundance and rates of
rBSC-1 protein synthesis and degradation to put rBSC-1 transport
activity at the appropriate level. Acid incubation did not alter the
decrease in rBSC-1 mRNA abundance during the first phase of cell
adaptation, and thus increased rBSC-1 mRNA abundance between the
9th and 16th hours of incubation (Fig. 9).
|
Studies after in Vivo CMA: Effects of in Vivo CMA on rBSC-1 Expression
After 6 days of NH4Cl administration (8-11 mmol/day),
rats had hyperchloremic metabolic acidosis accompanied with a small decrease in plasma potassium concentration that did not reach statistical significance (Table IV).
Plasma sodium concentration was unchanged, but plasma osmolality was
significantly increased during CMA (Table IV); these results indicate
that the increase in plasma osmolality was due to unidentified neutral
solute(s) and thus may not have corresponded to an effective
hyperosmolality. As seen in a weight gain lower in acidotic than in
control rats (30 ± 11 g versus 54 ± 8, respectively; p < 0.01), CMA probably caused a
moderate extracellular fluid volume contraction, as expected. As
assessed in crude membranes from MTAL primary suspensions, CMA
increased the rBSC-1 protein abundance by ~168% (p < 0.01) but did not affect that of -actin (Fig.
10). The rBSC-1 mRNA abundance was
also enhanced in MTALs from 4.6 ± 0.2 amol/100 ng of
RNAtot in control to 9.5 ± 1.1 in acidotic rats
(p < 0.02; Fig. 10), which represented a ~107%
increase.
|
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To induce metabolic acidosis with a different experimental protocol and to assess the time course of the CMA effects on rBSC-1 expression, rats were rendered acidotic by peritoneal dialysis. At the end of peritoneal dialysis (45 min), a pronounced metabolic acidosis was present in rats dialyzed against a bicarbonate-free NH4Cl-containing dialysate (Table IV). Three hours after the onset of peritoneal dialysis, hyperchloremic metabolic acidosis was still present and accompanied with a significant increase in plasma potassium concentration, as expected during the first hours of rapidly induced metabolic acidosis (Table IV); there was no difference in plasma osmolality and sodium concentration between control and experimental rats (Table IV). At this relatively early time (3 h), rBSC-1 mRNA abundance in MTAL primary suspensions was moderately but significantly enhanced by ~30% (p < 0.04; Fig. 11), but rBSC-1 protein abundance was not yet increased in crude membranes from MTAL primary suspensions (Fig. 12). One or 2 days after onset of dialysis, metabolic acidosis was no more evident in the blood (Table IV) despite maintenance administration in drinking water of moderate amounts of NH4Cl (1.5-2 mmol/day) to experimental rats versus NaHCO3 to control rats; at this time, there was also no difference in body weight and in plasma osmolality and sodium, potassium, and chloride concentrations (Table IV). However, increases in rBSC-1 mRNA (~58%; p < 0.001; Fig. 11) and protein (~150%; p < 0.007; Fig. 12) abundance were noted in MTALs of rats given NH4Cl. These results indicate that the latter rats were well adapted to the acid load with respect to MTAL rBSC-1 expression.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
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Present results demonstrate for the first time that a long term
in vitro incubation of freshly harvested MTALs in an acid medium increases transport activity and protein and mRNA abundance of rBSC-1, the apical
Na+-K+(NH4+)-2Cl
cotransporter of the rat MTAL. Results obtained with actinomycin D and
cycloheximide strongly suggest that these effects resulted, at least in
part, from increased gene transcription and protein synthesis. In
vivo CMA also increased rBSC-1 expression in rat MTALs at the
protein and mRNA levels.
We have previously established that rat MTALs incubated for up to
16 h in HDMEM in a rotary shaking water bath, a preparation we
have named MTAL shake suspension, remain as well differentiated tubular
fragments in which apical
Na+-K+(NH4+)-2Cl
cotransport and Na+/H+ antiport (NHE-3) are
functionally present (24). This is confirmed in the present study in
which rBSC-1 protein and mRNA were detected in this preparation. We
have shown previously that
Na+-K+(NH4+)-2Cl
cotransport, Na+/H+ antiport, and
Na+/K+-ATPase activities were not appreciably
altered after 16 h of incubation as compared with the activities
observed in primary suspensions (24). In the present study, we have
observed that the rBSC-1 mRNA abundance decreased during the first
hours and then was stable from the 9th to 16th hour of incubation (Fig. 9). Taken together, these results suggest that freshly harvested MTAL
cells adapt to the new hormone-free in vitro environment with respect to rates of transcription and/or mRNA degradation and
mRNA translation efficiency to maintain appropriate levels of
rBSC-1 protein abundance and transport activity. Thus, the MTAL shake
suspension is made of freshly harvested MTALs on which long term
in vitro studies may be conducted. This is of importance because MTAL cells in culture usually lack appropriate differentiation and regulated MTAL apical carriers, as discussed previously (21, 24);
to our knowledge, one of the rare preparations that express BSC-1 is a
cell culture line obtained from a transgenic mouse, and in which the
level of expression of BSC-1 is considerably less than in native TAL
cells (20).
As a result of a long term incubation in an acid HDMEM designed to
simulate metabolic acidosis,
Na+-K+(NH4+)-2Cl
cotransport activity increased by ~106% in intact MTAL cells and by
~35% in a membrane fraction enriched 7 times in MTAL apical membrane; it is possible that this quantitative difference may have
been explained by some stimulatory mechanism operational in intact
cells but lost during the fractionation procedure. In any case, the
acid-induced stimulation resulted at least in part from an increased
number of
Na+-K+(NH4+)-2Cl
cotransporters because the rBSC-1 protein abundance was augmented by
~46% in MTAL membranes, which was accompanied by a ~57% increase in rBSC-1 mRNA abundance; the increase in rBSC-1 protein abundance was observed in both apical membrane-enriched and crude MTAL membranes. The effect of acid incubation on rBSC-1 mRNA abundance occurred under the present experimental conditions between the 9th and 16th hour
of incubation, times at which control rBSC-1 mRNA abundance had
reached a new steady level; thus, it seems that a 7-h incubation time
was sufficient for acid incubation to increase rBSC-1 mRNA expression in MTAL shake suspensions. These effects did not result from
a nonspecific general stimulation of MTAL cellular processes since
alkaline phosphatase and Na+/K+-ATPase enzyme
activities and -actin protein abundance were not increased; the
intracellular buffering power was also not affected by acid incubation.
In this regard, it is known that in vitro acid incubation
does not induce cell hypertrophy (31). That Na+/K+-ATPase activity was not affected by acid
incubation in the present study is consistent with the lack of effect
of in vivo acidosis on MTAL
Na+/K+-ATPase activity (32).
The physiological significance of these in vitro findings was demonstrated by the observation that rBSC-1 expression in MTALs was stimulated by in vivo CMA. Indeed, rBSC-1 protein and mRNA abundances were both enhanced by CMA induced by two different experimental protocols, i.e. administration of important amounts of NH4Cl during 6 days or peritoneal dialysis against a bicarbonate-free and NH4Cl-containing dialysate followed by administration of moderate amounts of NH4Cl. The latter protocol allowed us to observe that rBSC-1 mRNA abundance was augmented after only 3 h of metabolic acidosis but that this was not yet accompanied with an increase in rBSC-1 protein abundance; thus, up-regulation of rBSC-1 mRNA preceded that of rBSC-1 protein. After 1 or 2 days of acid loading, both rBSC-1 mRNA and protein were up-regulated. It should be noted that up-regulation of rBSC-1 expression was observed whether acid loading caused a decrease or an increase in plasma potassium concentration, an increase or no change in plasma osmolality, and a decrease or no change in extracellular fluid volume as reflected by differences in weight gain (Table IV), depending on the duration of CMA and the way by which it was induced (NH4Cl administration in drinking water versus peritoneal dialysis). Results obtained in vitro in the present study point to an important role for an acid extracellular/intracellular pH.
Up-regulation of rBSC-1 was explained by stimulation of gene
transcription and protein synthesis because stimulation of
Na+-K+(NH4+)-2Cl
cotransport activity by in vitro acid incubation was
abolished by actinomycin D or cycloheximide. However, other
acid-invoked intracellular events, such as reduced rBSC-1 mRNA
decay or alteration in mRNA translation efficiency and membrane
trafficking of rBSC-1 protein, cannot be excluded. It should also be
noted that several alternatively spliced BSC-1 isoforms have been
described (14-16, 23); the rBSC-1 protein and mRNA determination
methods employed in the present work were both designed to detect
determinants common to the published rBSC-1 isoforms. Further work is
thus needed to test whether the stimulated BSC-1 expression we observed was due to an increase in one particular rBSC-1 isoform or to a global
increase in all isoforms.
The intracellular mechanisms by which acid incubation could have
stimulated rBSC-1 gene transcription were not investigated in the
present study. Previous studies in cultured renal cells have shown that
in vitro acid incubation activates, through intracellular acidification, the c-Src nonreceptor protein-tyrosine kinase, which
would constitute the first step of a signaling cascade stimulating expression of immediate early genes (c-fos,
c-jun, junB, and egr-1). Activation of
these transcription factors may in turn be responsible for many
cellular events, including stimulation of NHE-3 gene transcription (33,
34). Whether such a process might account for our findings observed in
freshly harvested MTALs remains to be established, since this mechanism
seems to be tissue-specific (33). In other tissues, incubation in acid
media inhibits expression of egr-1 in cultured osteoblasts
(35) and of 2-adrenergic receptor gene in cultured
smooth muscle cells (36). Moreover, the mBSC-1 promoter (20), unlike
that of NHE-3 (37), does not contain consensus binding sites for the
egr family of transcription factors.
In summary, present results establish for the first time that in
vivo CMA as well as long term acid incubation of freshly harvested
MTALs stimulates rBSC-1 expression in rat MTAL, as manifest by an
acid-induced increase in rBSC-1 mRNA, protein, and transport activity. Since the MTAL
Na+-K+(NH4+)-2Cl
cotransporter is a major MTAL apical
NH4+ carrier, these observations may
explain the adaptive increase in MTAL
NH4+ absorption in response to in
vivo metabolic acidosis. Indeed, Good (5) has shown that
NH4+ absorption increases by 37% in rat
MTALs isolated and perfused in vitro in response to CMA of
5-8 days' duration. Our results suggest that metabolic acidosis
per se, among other possible stimuli during in
vivo CMA, stimulates the expression of BSC-1 probably via effects
of an acid intracellular pH to cause the adaptive increase in MTAL
NH4+ absorption. As mentioned above, the
increased ability of the MTAL to absorb ammonia would aid in enhancing
ammonium excretion and thus would contribute to the renal response
against CMA. It is worth noting that, if CMA also stimulates BSC-1 in
the cortical TAL, sodium chloride absorption should be enhanced along
the entire TAL, which would limit the renal sodium wasting seen during
chronic metabolic acidosis due to decreased NaCl absorption in the
proximal tubule (38).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Pascale Borensztein and Zoubida Karim for helpful technical advice in RT-PCR and immunoblotting designs, respectively, and Laetitia Micheli for technical help.
| |
FOOTNOTES |
|---|
* This work was supported in part by grants from INSERM, the Université Paris 6, the Fondation pour la Recherche Médicale Française, the Fondation de France, and the National Institutes of Health.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.
¶ Supported by National Institutes of Health Physician Scientist Award DK02103.
Supported by National Institutes of Health Grant DK36803.
** To whom correspondence should be addressed. Present address: INSERM Unité 426, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75870 Paris cédex 18, France. Tel.: 33-1-44-85-62-81; Fax: 33-1-42-28-15-64; E-mail: bichara{at}bichat.inserm.fr.
The abbreviations used are: CMA, chronic metabolic acidosis; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; ANOVA, analysis of variance; BCECF-AM, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; bp, base pair(s); cRNA, complementary RNA; cDNA, complementary DNA; HDMEM, 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium; MTAL, medullary thick ascending limb; NHE, Na+/H+ exchanger; pHi, intracellular pH; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RNAtot, total RNA; RT, reverse transcription; TAL, thick ascending limb; TBS-T, Tris-buffered saline with Tween 20.
2 The public domain NIH Image program was developed at the United States National Institutes of Health and is available via the World Wide Web (http://rsb.info.nih.gov/nih-image).
3 Electron microscopy was performed by Gerard Feldmann and Alain Moreau, INSERM U327, Paris, France.
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Discussion
References
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Z. Karim, A. Attmane-Elakeb, V. Sibella, and M. Bichara Acid pH Increases the Stability of BSC1/NKCC2 mRNA in the Medullary Thick Ascending Limb J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2229 - 2236. [Abstract] [Full Text] [PDF]
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T.-H. Kwon, J. Nielsen, Y.-H. Kim, M. A. Knepper, J. Frokiaer, and S. Nielsen Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II Am J Physiol Renal Physiol, July 1, 2003; 285(1): F152 - F165. [Abstract] [Full Text] [PDF]
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M. Gallazzini, A. Attmane-Elakeb, D. B. Mount, S. C. Hebert, and M. Bichara Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb Am J Physiol Renal Physiol, May 1, 2003; 284(5): F977 - F986. [Abstract] [Full Text] [PDF]
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 J. N. Lorenz, N. R. Baird, L. M. Judd, W. T. Noonan, A. Andringa, T. Doetschman, P. A. Manning, L. H. Liu, M. L. Miller, and G. E. Shull Impaired Renal NaCl Absorption in Mice Lacking the ROMK Potassium Channel, a Model for Type II Bartter's Syndrome J. Biol. Chem., September 27, 2002; 277(40): 37871 - 37880. [Abstract] [Full Text] [PDF]
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M. Soleimani, T. Greeley, S. Petrovic, Z. Wang, H. Amlal, P. Kopp, and C. E. Burnham Pendrin: an apical Cl{-}/OH{-}/HCO3{-} exchanger in the kidney cortex Am J Physiol Renal Physiol, February 1, 2001; 280(2): F356 - F364. [Abstract] [Full Text] [PDF]
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A. Attmane-Elakeb, H. Amlal, and M. Bichara Ammonium carriers in medullary thick ascending limb Am J Physiol Renal Physiol, January 1, 2001; 280(1): F1 - F9. [Abstract] [Full Text] [PDF]
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A. Attmane-Elakeb, V. Sibella, C. Vernimmen, X. Belenfant, S. C. Hebert, and M. Bichara Regulation by Glucocorticoids of Expression and Activity of rBSC1, the Na+-K+(NH4+)-2Cl- Cotransporter of Medullary Thick Ascending Limb J. Biol. Chem., October 20, 2000; 275(43): 33548 - 33553. [Abstract] [Full Text] [PDF]
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