Parallel adaptation of the rabbit renal cortical
sodium/proton antiporter and
sodium/bicarbonate cotransporter in metabolic
acidosis and alkalosis.
T Akiba, … , V K Rocco, D G Warnock
J Clin Invest.
1987;80(2):308-315. https://doi.org/10.1172/JCI113074.
Recent studies have shown that the bicarbonate reabsorptive capacity of the proximal
tubule is increased in metabolic acidosis. For net bicarbonate reabsorption to be regulated,
there may be changes in the rate of apical H+ secretion as well as in the basolateral base
exit step. The present studies examined the rate of Na+/H+ exchange (acridine orange
method) and Na+/HCO3 cotransport (22Na uptake) in apical and basolateral membranes
prepared from the rabbit renal cortex by sucrose density gradient centrifugation. NH4Cl
loading was used to produce acidosis (arterial pH, 7.27 +/- 0.03), and Cl-deficient diet with
furosemide was used to produce alkalosis (arterial pH, 7.51 +/- 0.02). Maximal transport rate
(Vmax) of Na+/H+ antiporter and Na+/HCO3 cotransporter were inversely related with
plasma bicarbonate concentration from 6 to 39 mM. Furthermore, the maximal transport
rates of both systems varied in parallel; when Vmax for the Na+/HCO3 cotransporter was
plotted against Vmax for the Na+/H+ antiporter for each of the 24 groups of rabbits, the
regression coefficient (r) was 0.648 (P less than 0.001). There was no effect of acidosis or
alkalosis on affinity for Na+ of either transporter. We conclude that both apical and
basolateral H+/HCO3 transporters adapt during acid-base disturbances, and that the
maximal transport rates of both systems vary in parallel during such acid-base
perturbations.
Research Article
Find the latest version:
Parallel Adaptation
of the Rabbit
Renal
Cortical Sodium/Proton
Antiporter
and
Sodium/Bicarbonate Cotransporter
in
Metabolic
Acidosis
and Alkalosis
Takashi Akiba, VitoK.Rocco, and David G. Wamock
NephrologySection, VeteransAdministrationMedicalCenter, SanFrancisco, California94121; and CardiovascularResearchInstitute
and Department ofMedicine and Pharmacology, UniversityofCalifornia,SanFrancisco, California94143
Abstract
Recent studies
have shown that the bicarbonatereabsorptive
capacity
of the proximal
tubuleis increased in metabolic acidosis.For
netbicarbonate reabsorption
&o
beregulated,
there maybe
changes in
therateof
apical H+ secretion as well as in theba-solateral base exit
step.The
presentstudies examined
the rateof
Na+/H+
exchange
(acridine
orange method) andNa+/HCO3
cotransport
(22Na
uptake)in apical
andbasolateral
membranesprepared
from the rabbit
renalcortex
by sucrosedensity
gradientcentrifugation.
NH4C
loading
was used to produce acidosis(ar-terial pH, 7.27±0.03), and
Cl-deficient
diet withfurosemide
wasused
toproduce
alialosis
(arterial
pH,
7.51±0.02).
Maximaltransport
rate(V..)
of
Na+/H'
antiporter
andNa+/HCO3
co-transporter wereinversely related with plasma bicarbonate
con-centrationfrom 6
to39 mM. Furthermore, the maximal
transport ratesof both
systems
varied
inparallel;
when
V.
for the
Na+/
HCO3
cotransporter wasplotted against
V,,x
for
theNa+/H+
antiporter
for each of the 24
groupsof
rabbits, the regression
coefficient
(r)
was0.648
(P
<0.001).
There
was noeffect of
acidosis
oralkalosis
onaffinity for Na+ of either
transporter.We
conclude
that bothapical
and basolateralH+/HCO3
trans-porters
adapt during
acid-base
disturbances,
and that the
max-imal
transportratesof both
systems varyin
parallel during suchacid-base
perturbations.
Introduction
Reabsorption of
waterand solute by the renal
proximal tubule
is
adynamic process that
canadapt to several
changing
physi-ologic factors:
glomerular
filtration
rate, stateof acid-base and
electrolyte
balance,
and
different
hormones. The
Na+/H'
anti-porter
ofthe brush
border
membrane of
proximal
tubules shows
adaptive changes
in the
maximal
rateof transport
(V.,)
in
met-abolic acidosis
(1-3)
and
metabolic alkalosis (4).
Other
factors,
including
parathyroidectomy, glucocorticoid
administration,
unilateral nephrectomy,
potassium
depletion,
and renal
ablation
also
increase
the
V.,.
of
the
brush border
Na+/H+ antiporter
(1,
5-8).
Recently
asodium/bicarbonate
cotransporterhas
been
described in basolateral membrane vesicles
prepared
from rabbit
renal cortex
(9,
10). This transporter plays
asignificant role
inbicarbonate
reabsorption
and
regulation
of
intracellular pH in
Portions ofthese results have
been
reported inabstact
form(1987.
KidneyInt.31:404).
Addresscorrespondence and reprintrequests toD. G. Warnock, M.D.,
Department
ofMedicine
(111J),VeteransAdministrationMedicalCenter,4150ClementStreet, SanFrancisco, CA 94121.
Receivedforpublication8September1986and inrevisedform23 March 1987.
The Journalof ClinicalInvestigation,Inc. Volume80,August 1987,308-315
the
proximal
tubular cell(11-19).
Thefactors that regulate theactivity of this
transporter have not yet been identified. Recentwork
has
established
that systemic pH, andtherefore
peritubular
pH,
plays
acentral
rolein the regulation of
bicar-bonate
reabsorption
intheproximal tubule
(20, 21). Indeed,
theintrinsic
reabsorptive capacity
for
bicarbonate of the
proximal
tubule isincreased
inchronic respiratory (22) and
metabolicacidosis
(23), and decreased in chronic metabolic alkalosis (24).
Because
bicarbonate
reabsorption
represents avectorial
processby
which
H'
is secreted
acrossthe brush border membrane and
base
equivalents
aretransported
acrossthe basolateral
mem-brane, these
findings raise the
possibility
that the
activity
of the
bicarbonate
reabsorptive
processesin both membranes
maybe
adaptively
regulated
during
statesof
chronic acid-base
distur-bance.
Renal
proximal cells in
ratswith chronic metabolic acidosis
show
significant reductions in intracellular pH,
asestimated by
weak
acid
accumulation (25), 31P nuclear
magnetic
resonance(26),
andthe
contentsof citrate
and
alpha-ketoglutarate
(27).
Such
afall in intracellular pH
might
stimulate
H'
secretion
acrossthe brush
border membrane
by activation of
the
Na`/H+
anti-porter
(28).
Ifthe base exit
step acrossthe basolateral membrane
is also
stimulated in
acidosis,
then the
intracellular
pH of
prox-imal tubular cells would remain low
asa consequenceof the
parallel
changes
in the
mechanisms of
H'
transportacrossthe
brush border membrane
(e.g.,
Na+/H'
antiporter),
and base exit
across
the
basolateral membrane.
The
presentstudies examined the
possibility
that
adaptive
changes
of
these
transportersoccurin
responsetoacid-base
dis-turbances. The results of
ourpresentstudies indicate that V,,s
of
Na+/H'
antiporter
and
Na`/H;03
cotransporteradapted
in
parallel
tometabolic
alkalosis and
acidosis,
without
significant
changes in Michaelis
constants(K.)
for sodium. This
adaptive
response toacid-base disturbances would maintain intracellular
pH in
arelatively
narrowrangewhen the
rateof transcellular
bicarbonate
absorption
is
changed by
metabolic acidosis
oral-kalosis.
Methods
Experimentalmodelsweredevisedto
study
metabolic alkalosis and aci-dosis ofrelatively shortduration, e.g., 24-48 h. Apaireddesign was utilized because control rabbitscandisplay
afairly
wide range ofsystemicpH (2).Ineveryinstance,agroup ofexperimentaland controlanimals,
obtained in thesameshipmentfroma
single
vendor,
werestudiedatthesametime. Female New Zealand white
rabbits, weighing
1.7-3.0kg,wereused in these studies.
Arterial bloodwasobtained witha
heparinized
syringe
from the me-dialearartery andkeptonice untilanalyzed
withanABLIAcid-Base Laboratory(Radiometer A/S, Copenhagen,
Denmark).
Therestof the plasmasamplewasusedto measuresodium, potassium,
chloride,ureanitrogen,andcreatinine concentrations
by
standard methods. Metabolic alkalosis model. Fourtosixrabbits in eachexperiment
werefeda
bicarbonate-containing,
chloride-deficient diet(Teklad Diet,
Madison, WI).
Thecontrolrabbits received standardregular
rabbit chow. Both groupswerefed24 g of chow viagastric
tube twicedaily
for 2 d. Itwasnecessarytogavage the animals because theexperimental
animals wouldnot eatthechloride-deficientdietontheirown.Theexperimental
diet contained 7.8%bicarbonate,
<0.05% elementalchloride,
and 3.42kcal/g. The
experimental
rabbitsalsoreceived 40 mgfurosemidei.m. once ortwiceaday
for bothdays
that the dietwasadministered(4).
The controlrabbitsreceivedasimilar volume of normal salineintramuscu-larly.
Both groupswereallowedfreeaccessto water.Arterialbloodgasanalysis
wasdoneonthethirdmorning
andappropriate
rabbitswere selectedfor sacrificeand membrane vesiclepreparation.
Metabolicacidosis model.Foodwasremovedfromagroupof four rabbits in the
morning,
andthey
weregavaged
with 35 mlof0.5 Mammoniumchloridethat
morning
andin the afternoon.They
were al-lowedtodrinkwateradlib.butwerenotfedduring
theacidosisperiod.
The controlrabbitsweregavaged
with 35 ml of 0.5 M ammonium bi-carbonate in themorning
andevening,
andwereallowedfreeaccesstoregular
rabbit chow andwater.Arterialblood gasanalysis
wasdone the nextmorning,
andappropriate
rabbitswereselectedforsacrifice and membranevesiclepreparation.
Membrane
preparation.
Basolateralmembranevesiclesand brush border membranevesicles fromtherenal corticeswereprepared by
the methodreportedby
Ivesetal.(29).
Althoughthe marker enzymeen-richment forthebrush border membranes isnot as
high
aswiththe traditionalmagnesium-aggregation technique,
thisapproach
waschosen because both basolateral and brush border membranes could beprepared
simultaneously
andharvestedfromthesame sucrosedensity
gradients.
Inbrief,
two orthreerabbits ofthe alkalosis group and their controlsor theacidosisgroup and their controlsweredecapitated,
andtheirkidneys
wereflushedvia the renalarterieswith 30 mlofice-coldhomogenizing solution(50
mM sucrose; 10 mMHepes/Tris,
pH 7.5;5 mMEGTA/Tris,
pH 7.5).The corticeswerethenhomogenized
in 300 mlofthe samebuffer with a SorvallOmnimixer(Omni
Instruments, Newton,CT)
atfullspeed
for4 min. Thehomogenate
wascentrifuged
for15 min at4,500
rpm in the JA-20rotorofaJ2-21centrifuge
(Beckman Instru-ments,Inc.,Fullerton,
CA), (2,450 g).
The supernatantswerecentriftged
at
17,000
rpm for 30 min(35,000
g).
Thepellets
weresuspended
in thebuffer
by
passagethrough
a22-gauge
needle andcentrifuged
at20,000
rpm for60 min(48,400 g).
Thefluffy
upperlayer
of thispelletwas washedwith 20 mlof isotonic buffer(250
mMsucrose;10 mMHepes/
Tris, pH 7.5)
andcentrifuged
at20,000
rpm for 60 min. Thefluffy
layer ofthisfinalpellet
(P3-light)
wasbottomloaded undersucrosedensity
gradients
asdetailed below.Sucrose
density
centrifugation.
35 mllinear 35-48%(wt/vol)sucrosedensity
gradients containing
10mMHepes/Tris, pH 7.5,wereused.The finalpellets
(P3-light)
weremixed with72%sucrose(1:2),bottom loaded undersucrosegradients,
andcentrifuged overnight
at27,000rpm inaSW-28rotor
(g&,,
=100,000)
andanL8-70Multracentrifuge (BeckmanInstruments,
Inc.).Thegradients
werealiquoted
into3-mlfractions with aBuchler AutoDensi-FlowII(Haake-Buchler Instruments,Fort Lee,NJ),
andsimilarfractions fromparallel gradients
werepooledtogether. Maltase andNa+/K+-dependentATPaseactivitiesofeachfractionwere measured.Thetwo orthreefractionswith thehighestmaltaseactivityandthe
highest
activity
ofNa+/K+-dependentATPase were chosen as brush border membrane vesicles and basolateral membrane vesicles,re-spectively.
Thepooled
fractionswerewashedwith 10 mM Hepes/Tris,pH 7.5,and storedat
-700C
until transport assays were undertaken.Enzyme assays. Maltaseactivitywasdetermined by measuringthe
release ofglucosefrom maltose with the hexokinase reaction (30). Na+/
K+-dependent
ATPaseactivitywasassayedwith the coupled assay of Schoneret al. (31). Protein concentrationwas determined using the method ofLowryetal.(32).Na+/Hi
antiporter assays.Na+/H+ exchange rates were determinedusing
thefluorescence quenchingof acridine orange as previously de-scribed(33).
Thebrush border membrane vesicles were preloaded with ice-cold buffer(250
mMsucrose; 10mMHepes/Tris, pH 6.0)
andad-justed
toaprotein
concentration of 15-25mg/ml.
Thefluorescenceofacridine orange(excitation,493nm;emission,530nm)wasmeasured in athermoregulatedratiospectrofluorometer.The assay solution con-sisted of 6MMacridine orange, 250 mM sucrose, 10 mMHepes/Trisat pH 7.5 and 150mMN-methylglucamine gluconate.
Na+/H'
exchangerates weredeterminedintriplicate by the rate of the recovery of fluo-rescenceafter addition of sodiumgluconate givingfinal sodium
concen-trations of90, 50,25, 10,and 5 mM.Eadie-Hofsteeanalysis(34)was
usedtoobtain values for
V.n
andKm.Amiloride inhibitionwas deter-minedusing 100MM amiloride and 90 mM sodium in the external buffer and wasuniformly found to be similar in all groups: control acidosis, 43.6±6.3%inhibition; acidosis,43.5±2.4%inhibition;controlalkalosis,39.4±1.6% inhibition; and alkalosis, 40.3±5.1% inhibition.
Na+/HCO3cotransporter assays.
Na`/HCO3
cotransport ratesweremeasuredby isotopic sodiumuptake (9). Briefly, 22Na uptake was
mea-sured at250Cbyarapidfiltrationtechnique.The basolateral membrane vesicles were preloaded with 200 mM sucrose, 50 mM Hepes/Tris,pH
7.5, and 1 mM magnesium chloride. The uptake was initiated by
vor-texing5Mlof membranesuspensionin 25 Ml ofuptakemedia. The final sodium concentrationswerevaried between 4.2 and 42mM,andtriplicate
determinations were made at each so that Eadie-Hofstee analysis could be used to obtain values for
V.
andKm.The 2-suptakeperiodwasterminated by rapid mixture of 30 Ml of incubation mixture with 1.5 ml of ice-cold stopsolution (200 mM sucrose, 50 mM Hepes/Tris, pH7.5,
40 mM potassium sulfate), the mixture rapidly transferred to aprewetted
0.45 Mm filter(Millipore/Continental Water Systems, Bedford,MA),
andwashedwith an additional 3 ml of ice-cold stop solution. Filters were counted byscintillation spectroscopy. Differences between 22Na uptake in the presence of 21 mM bicarbonatecompared withgluconate
were used as theactivityof the Na+/HCO3 cotransporter (9).
Statistics. Kinetic parameters were obtained by linear regression analysesofEadie-Hofstee plots.Atotalof six sets ofanimals were studied in thealkalosismodel, and six sets in the acidosis model. Experimental animals and control animals were included in each set. The treatment ofthe animalsin eachpaired control and experimental set of rabbits was as similar aspossible. Rabbits from the same shipment were used for each set.Vesiclepreparationand transport assays were done
simul-taneouslyfor each set so thatpairedstatisticalanalysescould be used to
analyzethe results.Statisticalsignificanceof the differences in the paired
studieswasestimatedby pairedt testbetween the control and experi-mental resultsin each set. Results are expressed as mean±SEM.
Results
Dietary manipulation and/ordiuretics significantly influenced thepH and bicarbonateconcentration of arterial blood in aci-doticandalkalotic groups (Table I). In the acidotic series, arterial blood pH was 7.27±0.03 in the experimental group and 7.41±0.02 inthe control group for this series. In the alkalotic series, arterial blood pHwas7.51±0.02 in the treatedgroupand 7.39±0.02inthecontrols. Bloodbicarbonate concentrations in theacidotic series were 9.7±1.1 mM and 22.0±0.7 mM in their controls.Bloodbicarbonate concentrations in the alkalotic series were
32.7±1.5
mM in the alkaloticgroup and 21.6±1.3 mM in theircontrols. The different sham treatments of both control groupsdid
notinduce
anydifferences
in their measured blood chemistries (Table I; nonpaired t tests). Acidotic rabbits hadsig-nificant
hypocapnia andhyperchloremia. ALkalotic rabbits hadsignificant
hyponatremia, hypochloremia, and hypokalemia. There weresignificant increases in the blood urea nitrogen levels in the alkalotic and acidotic groupscompared
with their controls, butthechanges
inthe serumcreatinine
concentrations did not reachstatistical
significance. These results suggest that both models of acid-base change were associated with volumede-pletion.
The
enrichments of maltase
activity
in
the brush borderTableI. Principal BloodParameters in Acidosis and Alkalosis
pH Pco2 HCO3 CI- Na K+ BUN Creatinine
U torr mM mM mM mM mg/dl mg/dl
Acidosisseries
Control 7.41±0.02 34.8±0.6 22.0±0.7 102.8±0.5 141.0±0.8 4.6±0.3
15.3±0.8
0.82±0.11
Acidosis 7.27+0.03 22.3±2.5 9.7±1.1 112.9±2.2 141.9±1.5 4.2±0.121.2±1.3
0.85±0.06
Mean
paired
delta -0.15+0.03*
-12.5±2.4*-12.3+1.3*
10.2±1.9* 0.9±1.5 -0.4±0.2 6.3±2.0*0.03±0.11
Alkalosisseries
Control 7.39±0.02 35.8±2.2 21.6±1.3 103.2±1.1 141.6±1.1 5.0±0.3
12.5±1.4
0.74±0.10
Alkalosis 7.51±0.02 40.1±0.9 32.7±1.5 85.7±2.0 132.4±1.2 3.5±0.1
34.8±4.4
1.14±0.12
Meanpaired delta
0.13±0.031
4.3±2.5 11.7±2.1*-17.5+2.2*
-9.2+0.8*
-1.5±0.4
22.3±3.8*
0.40±0.15
Mean±SEM. n=6 pairedsets of rabbits in each series. *P < 0.005. $P < 0.05(Only five sets of values were obtained for BUN and
creati-nine.) P<0.01.
Table IL Marker EnzymeAnalysesinAcidosis and Alkalosis
Basolateralmembrane Brush bordervesiclemaltase Na+/K' dependentATPase
SA EF SA EF
pmol/mg-min pmol/mg.min pmol/mgminr pmol/mg.min Acidosis series
Control 41.6±1.4 6.7±1.4 1.43±0.29 10.1±1.5
Acidosis 57.4±7.9 7.7±1.4 1.47±0.44 8.3±1.0
Meanpaired delta -15.5±8.5 -1.0±1.8 -0.04±0.22 1.8±1.5
Alkalosis series
Control 38.1±5.8 6.6±1.4 1.46±0.15 8.2±1.9
Alkalosis
33.4±4.4 5.8±0.9 1.56±0.29 6.3±0.9Meanpaireddelta 4.7±5.7 0.8±1.1 -0.10±0.29 1.9±1.4
Sixsetsofrabbitsin each series.
SA,
specific activity;
EF,
enrichment factor.membrane
vesicle
preparations compared with
the homogenateactivities
were7.7±1.4,
6.7±1.4, 5.8±0.9,
and
6.6±1.4 in the
acidosis
groupand
their controls,
and the
alkalosis
groupand
their
controls,
respectively. Enrichments of
Na+,K+-dependent
ATPase
activities
in the
basolateral
membrane
preparations
were8.3±1.0, 10.1±1.5,
6.3±0.9,
and
8.2±1.9
in the
acidosis
groupand
their controls, and in the
alkalosis
groupand
their controls,
respectively.
The
specific activities
and
enrichment
factors
for
both membrane
markers
werenotsignificantly different by paired
analysis
amongthe
various
groups.These data
arepresented
in
Table II. In
addition,
the
specific activities
werealso
the
samefor
bothmarkers in
thehomogenates of
all groups(data
notshown). Furthermore, there
werenotany significantcorrelations
between
the
enrichment
factors for either
enzymeand the blood
bicarbonate
concentrations
for
all
of
the 24
setsof experimental
and
control
animals
(data
notshown).
Kinetic
parametersof H+/HCO3
transporters aresumma-rized in Table
III.The
Kmvalues for sodium of the
Na+/H+
antiporter
of brush border membrane vesicles
were notsignifi-cantly different between
acidosis, aLkalosis,
and their
respective
controls.
Inaddition,
all
groupsof brush border membranes
demonstrated
identical degrees
of inhibition
of Na+/H+
anti-porter
activity
when tested
with 90
mMNaand 100 MM
amil-oride
(data
notshown).
In contrast,the
V.
of
theNa+/H+
antiporter
wasincreased
to6.75±1.12
arbitrary
fluorescence
units
(FU)'/mg
protein
* sin
acidosis compared
with 3.95±0.94
FU/mg
protein
sin the
simultaneous
control
group(mean
paired
difference,
2.81±0.33
FU/mg
protein
s; P<0.001).
V.
of the
Na+/H+
antiporter
in
the
alkalosis series
wasnotsignifi-cantly different
from that oftheir controls
(Table
I).
Inaddition,
there
were notanysignificant
correlations between marker
en-zyme
specific
activities (Table II) and
maximal transport ratesfor either the
Na/H antiporter
orthe
Na/HCO3
cotransporter(data
notshown),
ruling
outthe
possibility
that the
changes
in
V.
weredue
torelative differences
in
amountsof
brush border
or basolateral
membranes in
thedifferent
preparations.
Similarly,
the
Kms
for sodium ofthe
Nae/HCO3
cotransporter
in the
acidosis
and alkalosis
groupswere notsignificantly different
from
their controls.
Furthermore,
there
were notany significantcorrelations between the
Kms
of either
transportsystem and theblood bicarbonate concentrations for
all of the 24setsofexper-imental and
control animals
(data
notshown).
TheV.
of theNa+/HCO3
cotransporterin the
acidosis
group wasincreased
significantly
to3.56±0.55
nmol/mg protein
* 2scompared
with1.84±0.36
nmol/mg protein
* 2sin the vesicles from their control1. Abbreviation usedinthispaper:
FU,
arbitrary
fluorescence unit.TableIII.Kinetic Parameters of
H'
Transport Systems in Acidosis and AlkalosisNa+/H+antiporter Na+/HCO3cotransporter
V.sa K. V. K.
FU/mgs mM nmol/mg-2s mM
Acidosisseries
Control 3.95±0.94 15.3±0.8 1.84±0.36 9.8±1.4
Acidosis 6.75±1.12 23.6±4.6 3.56±0.55 14.3±2.9
Meanpaired delta 2.81±0.33* 8.3±4.6 1.71±0.51I 4.5±1.7
Alkalosisseries
Control 5.21±1.09 21.4±2.1 2.07±0.19 11.9±2.1
Alkalosis 3.55±0.74 17.8±1.9 1.14±0.18 9.0±1.7
Meanpaired delta -1.66±0.73 -3.6±1.9
-0.93±0.26*
-2.9±3.3Paired, two-tailed Studentt test.Sixpairedsetsof rabbits ineachseries. * P<0.001. tP<0.025.
rabbits (mean paired difference, 1.71±0.51 nmol/mg protein *2 s;P<0.025). Furthermore, Vn=of the
Na+/HCO3
cotransporterinthe alkalosisgroupwasdecreased significantlyto 1.14±0.18 nmol/mg proteins 2scompared with 2.07±0.19 nmol/mg pro-tein* 2s in their control group (mean paired difference, -0.93±0.26nmol/mgproteins 2s;P<0.025).
These datawere plotted as afunction of the blood bicar-bonate concentration in Figs. 1 and 2. Vms of the
Na+/H+
antiporterweresignificantly correlated with the blood
bicarbon-ateconcentration (r= 0.520; P<0.01; d.f., 22), withalinear regression equation ofy=-0.15 (±0.05)x+8.17 (±2.25).
Vmas
oftheNa+/HCO3cotransporterwerealsosignificantly correlated with blood bicarbonate concentration (r = 0.781; P<0.001;
d.f., 22), withalinearregression equation ofy=-0.11
(±0.02)x
+4.48(±0.77).
Thedata in Figs. 1 and 2represent all 24setsof
determi-nations, but the pairedresponses in the alkalosis and acidosis groups arenot apparent.Fig. 3presentsthepairedcomparisons
ofNa+/HCO3cotransporteractivity between the acidosis series
12
10
Ir-o a 8
c
0._
Z
< ,° 6
I CL
0 E
z D 4
0 _
zE 2
0
(squares) and the alkalosis series (circles). Theopensymbols for each seriesrepresenttheconcurrentcontrols, and the solid lines
connecttheappropriate controlmeasurements with the
exper-imentalmeasurementsmadeinthepairedgroupsof rabbits. In
everyinstance, there was anincreasein the
V..
of theNa+/ HCO3 cotransporter inacidosis compared with controls, and alsoadecreaseinV.
inthe alkalosis series. Theaveragevaluesandmeanpaired differencesarepresentedinTable III. Fig. 4presentsthepaired comparisons of
Na+/H'
antiporter activity between the acidosis series (squares) and thealkalosisseries(circles).Theopensymbolsfor each seriesrepresentthe
concurrentcontrols, and the solid linesconnecttheappropriate control measurements with the experimental measurements
made in thepairedsetofrabbits. Ineveryinstance, therewas anincrease in
V.
of theNa+/H'
antiporter in acidosis com-pared with controls. The decrease in activity in the alkalosisserieswasnotasstriking.Theaveragevalues andmeanpaired differencesarepresented in Table III.
Fig.
5 presentsacomparison
of the maximaltransportrates6 1
IAJ w 0: 0.
Z "
0 2.
t 06
Z c
w
0
a1
4 8 12 16 20 24 28 32 36 40
PLASMA BICARBONATECONCENTRATION (mM)
Figure1.Relationship betweenbloodbicarbonateconcentration and
maximalrateofNa+/H'antiporter.
V.
(FU/mgprotein s)wasde-terminedby Eadie-Hofstee analysis ofNa+/H'exchangeratesin brush bordervesicles. Each datapointrepresentsthe value obtained froma
singlemembranepreparation ina groupoftwoorthree rabbits.The
bicarbonateconcentrationwasobtainedbyarterialbloodgasanalysis
andrepresentsthemeanvalue for rabbits inthatgroup.TheV,,sof
theNa+/H'antiporterweresignificantlycorrelated withblood bicar-bonateconcentration(r=0.520;P<0.01; d.f., 22), withalinear
regression equation (solid line)ofy=-0.15(±0.05)x+8.17 (±2.25).
-4 8 12 16 20 24 28 32 36 40
PLASMABICARBONATE CONCENTRATION (mM)
Figure2.Relationshipbetweenblood bicarbonate concentrationand maximalrateofNa+/HCO3cotransporter. V, (nmol/mg protein'2
s)wasdeterminedbyEadie-Hofsteeanalysis of Na+/HCO3cotransport
ratesinbasolateral membranevesicles. Each data pointrepresentsthe
value obtained fromasinglemembrane preparation ina groupoftwo
orthree rabbits.Bicarbonateconcentrationwasobtainedbyarterial
bloodgasanalysisandrepresentsthemeanvaluefor rabbits inthat group.TheVimsweresignificantly correlated withbloodbicarbonate
concentration(r=0.781;P<0.001; d.f., 22), withalinear regression
equation (solid line)ofy=-0.11 (±0.02)x+4.48 (±0.77).
ParallelAdaptation ofRenal Hi/HCO3 Transport 311
A
5
a
4 a
3 aA
2 a
.
.
,
1
I12
10
8
c
._
°% 6
c.
"'4
U.
2
4 8 12 16 20 24 28 32 36 PLASMA BICARBONATE CONCENTRATION (mM)
WJ
I- 5
cr.N
zcr
8 3.
Z X
To E
0 2 4 6 8 10
V,,,OF Na/H ANTIPORTER (FU/mg protein/s) 40
Figure3.Relationship between blood bicarbonate concentration and maximal rate ofNae/HCO3cotransporter.Each datapoint represents the valueobtained from a single membrane preparation in a group of
two orthreerabbitsin theacidosisseries (squares) and in the
alkalosis
series(circles). Control studies(opensymbols)areconnected to the paired experimental group(closed symbols).
for both the
Na+/H'
antiporter and Na+/HCO3
cotransporterin
alkalosis
(circles) and acidosis (squares). The solid lines
con-nect eachexperimental point with
the concurrent controlpoint.
Two
important points
emergefrom this figure.
(a) There was aconcordance between the maximal
ratesfor
both
transport sys-tems,and
(b)
there
was astriking parallelism
in the
adaptive
changes in
V.
for both
transport systemsin
response toacidosis
(closed squares)
and
alkalosis
(closed circles).
The
regression
line
for all of the data
points (dashed line)
is also included in
Fig.
5(y
=0.31
[±0.08]x
+0.66
[±0.94]).
This
regression
wassignif-icant (P
<0.001;
d.f., 22) with
an rvalue
of 0.648.
Discussion
Technical
issues. There are at least four distinct
H+/HCO3
transportmechanisms
in the
apical
and basolateral
membranes
of the
proximal
tubule. The
Na+/H'
antiporter
and
Na+/HCO3
cotransporterarethe
major
systemsand
werethe
focus of the
V-4 8 12 16 20 24 28 32 36 PLASMA BICARBONATE CONCENTRATION(mM)
40
Figure4.Relationshipbetweenblood bicarbonate concentration and
maximalrateofNa+/H'antiporter.Each datapointrepresents the
value obtained fromasinglemembranepreparationinagroupoftwo
orthree rabbitsintheacidosis series(squares)and in the alkalosis
se-ries(circles). Control studies (open symbols)areconnectedtothe
paired experimentalgroup(closed symbols).
12
Figure 5.Relationship between maximal rate of the Na+/HCO3 co-transporter and that ofthe
Na+/H1
antiporter. Each data point repre-sentsthe valueobtained from a single membrane preparation in a groupof two or three rabbits in the acidosis series (squares) and in the alkalosisseries(circles).Control studies (open symbols) areconnected tothepaired experimentalgroup(closed symbols).Regression linefor allofthe datapoints is shown as a dashedline(y=0.31 [±0.081x+0.66[±0.94]). This regressionwassignificant (P < 0.001; d.f., 22) with an r value of 0.648.
present
studies.
Itis
difficult
toquantitate the separatecontri-butions of
Na+/HCO3
cotransportand
HCO3/C1- exchange
tobicarbonate
exit
acrossthe basolateral membrane,
soweonly
measured the rate
of Na+/HCO3
cotransportin the absenceof
any
chloride ions. Recent studies have reported that the Na+/
HCO3
cotransporter can bedemonstratedin
the complete ab-senceof chloride (9, 10). This approach does
notpreclude the
possibility
that
the
HCO3/Cr-
exchanger
might
also be
regulated
by
metabolic acidosis
oralkalosis,
eventhough
this
transport systemis
thought
toplay
arelatively minor role
inbicarbonate
exit
acrossthe
basolateral membrane
(35).
At present,the
con-tribution of
anapical
membrane
H+-ATPase
tobicarbonate
reabsorption
in the
proximal
tubule
is
notwell
defined (20, 36),
so
this
transport system was notexamined in the present studies.
The
presentexperiments utilized
sucrosedensity gradients
(29) for the simultaneous
preparation
ofbrush border and
apical
membranes
from
the samekidneys.
Although this
advantage
wasconsidered crucial for the
presentexperimental
design,
it
must
be
pointed
out that theresulting
brush border
membrane
vesicles do
nothave
the samedegree of enrichment
asis
obtained
with the standard
magnesium
aggregation
method in
this
lab-oratory
(2,
33)
because the
sucrosedensity preparation
has
beenoptimized
for the
preparation
of basolateral membrane vesicles
(29). Furthermore, the absolutes
ratesof
Na+/H'
antiporter
ac-tivity
compare verywell
with
previous
determinations in control
and
metabolic acidosis
(2),
lending
further
supporttothe ade-quacyof
themembrane
preparations
for
the presentstudies.
Another
point relates
to theassessmentof
theacid-base
statusof
theanimals.
We havepreviously
observed
thatthemeasure-ment
of arterial pH
atthe time of sacrifice
may underestimate theseverity
of
metabolic
acidosis, presumably
becauseof
acutehyperventilation.
In aprevious study,
weused
plasma
total
CO2
contents as
measured
by
microcalorimetry (2).
Inthe present
study,
weused arterial blood
gasdeterminations,
but found that the bloodbicarbonate concentration
wasthemostappropriate
variable for
estimating
theacid-base
statusbecause any short-termeffects of
hyperventilation
atthetime of
sampling
would beminimized.
312 T.Akiba, V.KRocco,andD.G. Warnock
w
I-0
a-z
x
N,
0 z LU. 0
E1
0 .
AM 5 0
0.~~ co
en"
z 4
I-.-to0 0..
L) E2 Z c
2 I
Experimental
models for metabolic acidosis and alkalosis
were chosen to represent more
than
an acutechange in
acid-base
status(Table I).
Atleast three animals
weretreated in each
group,
and
arterial blood
gasdeterminations
weredone
onthe
morning of
sacrifice and
membranepreparation. Two
orthree
animals
wereused
for the membrane
preparations,
the choice
being based
uponthe measured blood bicarbonate
concentra-tions.
Thus, the animals
wereselected before sacrifice
tomostnearly
representthe
desired acid-base condition. In
addition,
two orthree
control rabbits
weresimultaneously
sacrificed with
their
experimental
groupso as tominimize
spontaneousvari-ations in the membrane
transportrates.Our
previous
studies
have
demonstrated the absolute
necessity
of such
apaired design
(2). The open(control) symbols in
Figs.
3 and
4show the
fairly
wide
variability in
transport rates eventhough the acid-base
statusof the control animals
wascarefully
defined. While it is obvious
that there may
be
factors other
thanacid-base
statusthat
accountfor variation in basal
transport rates,the
presentstudies used
contemporaneous
controls in
aneffort to minimize intraanimal
variability.
Whereasboth
acidosis and alkalosis
wereassociated
with mild
volumedepletion
(judging from the increased blood
urea
nitrogen
levels), the volume
status wassimilar in both
setsof controls
sothis factor
may notnecessarily
explain
the
vari-ability
observed in the control
groups.A
final
technical point pertains
tothe
determination of
max-imal
ratesof
transport. Atleast
five sodium concentrations
were used ineach
assay,and each
wasassayed in
quadruplicate
sothat
Vm.
and Km values could be compared between
groups.While it
would bedesirable
to comparethe
ratesof
H'
secretion
across
the
apical membrane
tothe
rateof
bicarbonate exit
acrossthe
basolateral
membrane, such
acomparison
would be
pre-maturein
ourview.
Maximal
ratesof
transport aredetermined
under
different
experimental
conditions for each
transport sys-tem.The actual
ratesof
transportin
vitro
maywell be
affected
by the
intracellular pH,
buffering,
divalent
cations,
etc.Fur-thermore, it is
notcertain that the amiloride-sensitive
Na+/H'
antiporter is
theonly
meansby which
H'
is secreted
acrossthe
apical
membrane(20, 36).
Forthese
reasons, eventhough it is
possible
toconvert theobserved
ratesof
Na+/H'
exchange (FU/
mg
protein
*s)
toflux
rates(37),
separateunits
have beenmain-tained for
theNa+/H'
antiporter
andNa+/HCO3
cotransporter, andquantitative
comparisons of the
twoseparate transport sys-temshave
notbeen made.
Parallel adaptation of H+/HCO3
transporters. The
present
experiments
demonstrate that
Vm.
values
of Na+/H'
antiporter
and
Na+/HCO3
cotransporter are regulatedin
a parallelfashion
(Fig.
5). Bothsystems vary inversely with the blood bicarbonateconcentration (Figs. 1
and2).
Vm.,
of
the Na+/H+antiporter
and
Na+/HCO3
cotransporter increased in metabolic acidosis anddecreased in metabolic
alkalosis, without any change in theKm
values
of
either
system (Table III). The comparison inFig. 5emphasizes
the parallel changes that occurred in each system.Furthermore, the fact that the control points (open symbols) are
distributed
along the regression indicates that there is a strong concordanceof
the maximal transport rates for both systems invivo.
Most
of
thefiltered
loadof
HCO3 is reabsorbed in theprox-imal
tubule (20).The rate ofproximal tubular bicarbonatereab-sorption shows
strong dependence on the filtered load of bicar-bonate(22,
38, 39). In chronic metabolic acidosis induced by ammoniumchloride,
the absoluterateof total
CO2 absorption
in the
proximal
tubuleis reducedwhen
compared
with
controlhydropenic conditions (40). When compared at comparable
lu-minal total
CO2 concentrations and tubular fluid flow
rates,however, the
rateoftotal
CO2
absorption is significantly
increased
in the acidotic
state(23).
Therefore, whereas the increased
V,,of the
Na+/H'
antiporter in acidosis (1) would increase proximal
bicarbonate reabsorption, the absolute
rateof
reabsorption
ap-pears tobe limited in vivo by the reduction in bicarbonate
con-centration of the glomerular filtrate (23, 40). Even though
theabsolute
rateof
bicarbonate
reabsorption is reduced in vivo in
metabolic acidosis, it
appearsthat
the
adaptation of the
V..
of
the
Na+/H'
antiporter
is ofphysiologic importance.
The datain
Fig.
1confirm
ourprevious
finding that the degree of adaption
of
V.
is
directly related
tothe
severity of induced metabolic
acidosis.
Thedirect implication of this
finding is that the renal
responses to
perturbations in systemic pH and/or bicarbonate
concentration
areof
regulatory
importance, in
contrast topre-vious
interpretations
(41).Bicarbonate
absorption by
proximal
renal tubules is
consid-ered
tobe mediated by
Na+/H'
exchange
and perhaps
H+-de-pendent
ATPasein the
apical
membranes
(20, 36),
and Na+/
HCO3
cotransport (9-19) and14CO3/Cl-
exchange(35, 42) in
the
basolateral membrane. Of these
transporters,the
Na+/H'
antiporter
in the
brush border membrane and
Nae/HCO3
co-transporterin the basolateral membrane
areconsidered
toplay
major roles in
regulating
the
intracellular
pH
of
proximal
tubular
cells
(17).
Our
presentfinding,
that there is
parallel adaptation
of the
Na+/H'
antiporter and the
Na+/HCO3
cotransporterin
acid-base
perturbations,
suggeststhat
changes
inintracellular
pH
areminimized
during
systemic
acid-base
perturbations
asaresult
of this dual
regulation.
Alpern
and Chambers
(17) recently
demonstrated that the effect
onintracellular
pH
of basolateral
Na+/HCO3
exceeds that
of the
apical
membrane
Na+/H'
anti-porter
during
short-term
changes
in the
pH
of luminal and
peri-tubular
perfusion
solutions in studies of the
it
vivo
microper-fused
ratproximal
convoluted
tubule. Whether this
samerelative
importance
pertains tothe subacute
acid-base
changes
examined
in the
presentstudies
is
aquestion
that
warrants
direct
investi-gation.
Cohn
etal.
(1) observed that
Na+/H+ exchange
activity
wasincreased
in brush border
vesicles
prepared from acidotic dogs.
Tsai
etal.
(2) observed that the
V.
of
Na+/H'
antiporter
in-creased in renal
cortical brush border membrane vesicles
pre-pared from rabbits with metabolic
acidosis. This
finding
wasalso
observed in
ratsby Kinsella
etal.
(2). We confirmed
these
findings
in the
presentstudy, and have observed that
V.
of
the
Na+/HCO3
cotransporterin basolateral membrane
vesicles
is also
increased in metabolic acidosis. Such parallel regulation
may
be
amechanism
tomaintain
acid-base
homeostasisof
theanimal, with
maintenance of intracellular
pHin
arelatively
nar-rowrange.Another
possibility is
that cell pH falls duringmet-abolic acidosis and this in
turnstimulates
theactivity of theapical
membrane
Na`/H+
antiporter
(28). Concurrent activationof the basolateral bicarbonate exit
step wouldmaintain
therel-atively
acidic
cell pH and therebymaintain
the stimulation of theapical
H+secretory
system(s) inmetabolic acidosis.Alter-natively,
theincreased
V.
of theNa+/HCO3 cotransporter may be aprimary
eventin metabolic acidosis,
decreasing the cellbicarbonate
and pH, andthereby activating the Na+/H+ anti-porter(28)
sothat cell
pHbecomes
less acidic. This sequenceof
eventsis
consistent with themeasured rates of both transportsystems
during
short-term
changes
in the luminal
andperitubular
pH (17). However,
theadaption
tosubacute
acid-base
bations examined in our studies is not simply a matter of allo-stericactivation of the transporters, but rather an absolute in-crease in
Vm.
(either number of carriers or increased turnover of eachcarrier) which persists when the membranes are isolated and studied in vitro under defined conditions ("memory effect"). Whatever theteleologicreasonfor the parallel regulation ofbothtransportsystems during acid-base
disturbances,
it seems likely thatthecombined effect would maintain cell pH within arel-atively
narrow,albeit acidotic
range.At
the expense ofcontinued
intracellularacidosis, the secretion of
H'
into the lumen, and bicarbonatereabsorption would bemaximized.
Animplication
of this
hypothesis is that the
responseof
theproximal
tubule tometabolic acidosis
woulddefend systemic
pH by enhancing proximalproton secretion andbicarbonate
reabsorptiondespite continuedintracellular acidosis.
In contrast to
metabolic
acidosis, regulation
ofbicarbonate absorption in proximal tubules of alkalotic animals is somewhatcontroversial. Reduced
glomerular
filtration is
responsible, atleast acutely, for
maintaining
thealkalotic
statein
Munich-Wistar rats(43). Inlonger term studies, increased bicarbonate absorptionby
the renalproximal
tubule
wasobserved
even whenglomerular filtration rate was restored to normal (38). This increase wasaccounted for
by chronic adaptive hypertrophy of
theproximal
tubule and a
load-dependent
responseindistinguishable from
that
seenin normal
rats(38). Recently Liu and
Cogan (39)
found
that
bicarbonate
rebsorption in the late
proximal convoluted
tubule
wasflow dependent but also
inhibited
by alkalemia and
chronic metabolicalkalosis
(24).
This inhibition ofbicarbonate
absorption
wasattributed
toimpaired bicarbonate exit
from the
cell
(24,
38). Our results(Fig.
2and TableI)
suggestthat activity
of the
bicarbonate exit
stepis decreased
during metabolic
al-kalosis because
of
adecrease
inthe
maximal
activity of
the Na+/HCO3
cotransporter.Suspended
proximal
tubularcells from rabbits withmetabolic
alkalosis showed
adecrease
inamiloride-sensitive,
sodium-de-pendent
intracellular alkalinization
compared
tonormalcontrols
(4). The
presentstudies used
asimilar
metabolic alkalosis
pro-tocol,
asdid Blumenthal
et al.(4),
and demonstrated thatNa+/
HCO3
cotransportactivity
wassuppressed in metabolic
alkalosis.
The
model utilized
is
diuretic-induced, C--deficiency
alkalosis
with
attendant
hypokalemia.
The
fact that
Na+/H+
antiporter
activity
was notsignificantly
reduced
inthis model of
hypoka-lemic,
hypochloremic
metabolic alkalosis
maybe the result
of
hypokalemia,
which has been shown
to increasethe
activity ofthe
Na+/H'
antiporter (8).
Eventhough
reduction
inV,,.
ofthe
Na+/H+
antiporter
was notstatistically significant by paired
ttest(Table
III),
it
should
bepointed
outthatV.,
decreasedin
each
of the six
experiments,
but
the controlratesofNa+/H+
antiporter
activity
werequite
variable(Fig. 4).
Insummary, maximal transport rates
of
theNa+/H+
anti-porter and theNa+/HCO3
cotransporter areinversely
relatedwith the plasma bicarbonate concentration from 6
to39
mM.Furthermore, the maximal
transportratesof both systems varied inparallel;
whenVm.
for
theNa+/HCO3
cotransporter
wasplotted
against
Vm..
for
theNa+/H'
antiporter
for each of the 24 groupsof
rabbits,
the regressioncoefficient
(r)
was0.648(P
<0.001).
Therewaso
effIect of acidosisor alkalosis on theaffinity
for
Na+of either
transporter. We conclude that bothapical
andbasolateral
H+/HCO3
transportersadapt
during
acid-base
disturbances,
and that the maximal transportratesofbothsystems vary
in
parallel.
Theseparallel
changes
would maintain theintracellular
pH
inarelatively
narrowrangeduring
chronicchanges
in
the extracellularpHand couldrepresentanadaptive responseof
theproximal tubule that would tend to correct thechronic
acid-base disturbance.
Acknowledgments
Thisprojectwas supported by the Veterans Administration Research
ServiceandNational Institutes of Health grant AM-19407 (Dr. Warnock). Dr.Akiba and Dr. Rocco were post-doctoral fellows in theCardiovascular
Research Institute duringthesestudies. Dr. Akiba was supported in part by a stipendfrom the National Kidney Foundation. Dr. Rocco was sup-ported by National Institutes of Health Training GrantAM-07219.
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