Creatine Transport in Brush-Border Membrane Vesicles Isolated from Rat Kidney Cortex
MARTA GARCÍA-DELGADO,
MARÍA J. PERAL,
MERCEDES CANO,
MARÍA L. CALONGE and
ANUNCIACIÓN A. ILUNDÁIN
Department of Animal Physiology and Biology, Facultad de Farmacia,
Universidad de Sevilla, Sevilla, Spain.
Correspondence to Dr. Anunciación A.
Ilundáin, Departamento
Fisiología y
Biología Animal, Facultad de Farmacia, C)
Tramontana s/n, 41012 Sevilla, Spain. E-mail:
Ilundain{at}cica.es
Abstract. The kidney efficiently salvages creatine from theurine;
however, the mechanism(s) that mediates renal creatinereabsorption has not
been investigated. This study characterizesthe creatine transport mechanism
in brush-border membrane vesiclesisolated from the rat renal cortex. An
osmolality plot revealedthat creatine is transported into an osmotically
active spaceand that it is also bound to the membranes. An inwardly directed
NaClgradient stimulated creatine uptake and the time course of uptake
exhibitedan overshoot phenomenon, which indicates the presence of anactive
process for creatine in these membranes. The uptake ofcreatine showed an
absolute requirement for both Na+ and Cl-.The NaCl
gradient—dependent creatine uptake was stimulatedby a
valinomycin-induced, inside-negative, K+-diffusion potential,which
suggests that the uptake process is electrogenic. Stoichiometricanalyses
indicated a probable couple ratio of 2 Na+:1 Cl-:1
creatinemolecule. The kinetic study showed that creatine is transportedby a
high-affinity system (Km of 15 µM). Creatine uptakewas
inhibited by a 100-fold excess of various compounds withthe following potency
order: cold creatine = guanidinopropionicacid > nipecotic acid >
-aminobutyric acid (GABA) = ß-alanine= betaine, whereas
carnitine, glycine, taurine, and cholinewere without effect. This pattern of
inhibition differs fromthat observed for GABA uptake (unlabeled GABA = GPA
> ß-alanine> nipecotic acid >> creatine). The conclusion drawn
was thatthe apical membrane of the renal cortical tubules contains anactive,
high-affinity, electrogenic, 2 Na+/1 Cl-/creatine
cotransporter.
Phosphocreatine is an important energy transducer in heart,brain, and
skeletal muscle. These tissues, however, which arethe major consumers of
phosphocreatine, do not synthesize creatine—theyobtain it or its
immediate precursor (guanidinoacetic acid)from plasma.
Human and other mammals may obtain part of their creatine requirementby
dietary intake but are able, on a creatine-free diet, tocover it completely
by endogenous biosynthesis (1).
The kidneyefficiently salvages creatine from the urine, and adult humanmales
on a creatine-free diet usually excrete little creatine.However, notable
quantities of creatine are excreted under aninadequate diet, fasting, and
other muscle massreducing conditions
(1).
Recent studies have revealed that the human brain/spinal cordcreatine
transporter (CREAT) is a member of the super-familyof proteins that includes
the family of Na+ - and Cl- - dependenttransporters,
which are responsible for the uptake of certainneurotransmitters
(e.g., dopamine, -aminobutyric acid (GABA),serotonin, and
norepinephrine) and amino acids (e.g., glycine)
(2,3,4,5).
Withinthis family, the human CREAT is strongly related to a subfamilythat
includes the transporters of taurine, betaine, and GABA.The CREAT mRNA has a
wide distribution, being particularly abundantin the brain, spinal cord,
skeletal muscle, heart, male reproductivesystem, and kidney
(5,6,7,8,9,10,11,12).
In the kidney, theCREAT mRNA is evenly distributed between cortex and medulla
(11,12).
Plasma membrane creatine transport has been studied in skeletalmuscle
(13), cell culture
preparations
(14,15),
human monocytesand macrophages
(16), and astroglia-rich
cultures (17). Thesestudies
revealed that creatine is transported by an active and
Na+-dependentmechanism. In the liver, however, creatine is
transported byan active and pH-dependent mechanism
(18). Even though it isknown
that the kidney tubules reabsorb creatine
(19) and thatthe CREAT mRNA
is particularly abundant in the kidney
(11,12),
thefunctional characterization of the mechanism(s) that mediatesrenal
creatine reabsorption has not been addressed. The purposeof the present study
was to characterize the transport of creatinein brush-border membrane
vesicles (BBMV) isolated from the renalcortex of the rat.
BBMV Preparation
Male Wistar rats (250 to 300 g) were anesthetized with ether.The animal
experimentation was conducted in accordance withthe National Institutes of
Health Guide for the Care and Useof Laboratory Animals.
BBMV were isolated from the kidney cortex of male Wistar ratsfollowing the
method of Biber et al.
(20). Briefly, kidney cortex
slicesfrom one rat were homogenized in 15 ml of isolation buffer (inmM, 300
mannitol, 5 ethyleneglycoltetraacetic acid, and 12 Tris-HCl[pH 7.4]) with the
Ystral Polytron on the setting 5 for 2 min.Twenty-one milliliters of cold
bidistilled water and MgCl2,up to a final concentration of 10 mM,
were added to the homogenate.The suspension was gently stirred for 20 min and
then centrifugedat 1900 x g for 15 min. The resultant
supernatant was centrifugedat 30,000 x g for 30 min, and the
resultant pellet was resuspendedin 30 ml of 150 mM mannitol, 6 mM Tris-HCl
(pH 7.4), and 2.5mM ethyleneglycoltetraacetic acid and homogenized with a
glass-Teflonpotter. MgCl2, at final concentration of 10 mM, was
added tothe homogenate. The suspension was gently stirred for 20 minand then
centrifuged at 1900 x g for 10 min. The resultant supernatant
wascentrifuged at 30,000 x g for 30 min, and the resultant
pelletwas resuspended in 0.5 ml of the appropriate loading buffer.The
suspension was made homogenous by passing it through a 20-gaugeneedle several
times and diluted up to 30 ml of the loadingbuffer. The suspension was
centrifuged at 30,000 x g for 30min. The isolated apical
membranes were made homogenous by passingthem through a 25-gauge and a
28-gauge needle several timesand stored in liquid nitrogen until used. All of
the steps werecarried out at 4°C. Protein was measured by the method of
Bradford(21), with the use of
bovine serum albumin as the standard.Unless otherwise stated, the BBMV were
loaded with a pH 7.5buffer that consisted of (in mM) 500 mannitol, 100 K
gluconate,and 50 HEPES-Tris.
Uptake Studies
Either creatine or GABA uptake was measured by a rapid filtration
technique,as described previously
(22). Except where indicated
otherwise,the uptake buffer consisted of (in mM) 200 mannitol, 250 NaCl,50
HEPES-Tris (pH 7.5), 0.020 valinomycin, and either 5 µM
[14C]-creatineor 10 nM [3H]-GABA. The amount of protein
in the assay tuberanged from 100 to 150 µg/100 µ1 of uptake buffer.
Chemicals
[14C]-creatine (carrier free) and [3H]-GABA were
purchased fromAmersham (Madrid, Spain). The other compounds and salts used
wereobtained from Sigma Chemical Co. (Madrid, Spain).
Statistical Analyses
Individual experiments were carried out in triplicate. Dataare presented
as mean ± SEM for n separated BBMV preparations.In the
figures, the vertical bars that represent the SEM wereomitted when they were
smaller than symbol height. Comparisonbetween different experimental groups
was evaluated by the two-tailedt test.
Effect of Osmolarity on Creatine Uptake
The binding of [14C]-creatine to the membranes was calculated,
at120 min, as the uptake of creatine by the BBMV at infinite osmolarity,
i.e.,when there is no intravesicular space. Uptake of creatine in
renalBBMV was an inverse linear function of the extravesicular osmolarity
(Figure 1),which indicates
that creatine is transported into an osmoticallyactive space.
Figure 1. External osmolarity and creatine uptake into renal cortex brush-border
membrane vesicles (BBMV). Medium osmolarity was increased by the addition of
mannitol. A 5-µM creatine uptake was measured during 120 min. The
composition of the buffers is given in the Materials and Methods section. The
line was calculated by linear regression analysis. The number (n) of
separated BBMV preparations was 3.
Creatine Uptake versus Time
The time course of [14C]-creatine uptake into renal BBMV
revealedthat, in the presence of an inwardly directed NaCl gradientand an
inside negative electrical membrane potential, creatinetransiently
accumulates in the vesicular space (Figure
2), thepeak of uptake being at 1 min. No overshoot was observed
whenthe extravesicular NaCl was replaced isosmotically by K gluconate.In the
presence of NaCl, creatine uptake increased linearlyup to 20 s. On the bases
of these results, a 15-s incubationtime was adopted to determine the initial
rate of creatine uptakeinto BBMV.
Figure 2. Time course of creatine uptake into BBMV. A 5-µM creatine uptake was
measured in the absence (; NaCl isosmotically replaced by K gluconate)
and presence ([UNK]) of inwardly directed NaCl gradient as a function of time.
Other details as in Figure
1.
Specificity of Creatine Uptake into Renal BBMV
[14C]-creatine uptake in renal BBMV was measured in the absence
andpresence of the compounds listed in
Table 1. In any of the
concentrationstested, cold creatine and guanidinopropionic acid (GPA) were
themore potent inhibitors; GABA, ß-alanine, and betainehave a small
inhibitory effect on creatine uptake; and eithercarnitine, glycine, taurine,
or choline have, if any, a smallinhibitory effect on creatine uptake.
Table 1. Specificity of either creatine or GABA uptake in renal cortex
brush-border membrane vesicles (BBMV)
The rat kidney cortex BBMV also transiently accumulated GABAin the
presence of an inwardly directed NaCl gradient (datanot shown). The effects
of several modifiers on the initialrate of GABA uptake are shown in
Table 1. The more potent
inhibitorsof GABA uptake were cold GABA, GPA, and ß-alanine.Nipecotic
acid, described as an inhibitor of the neuronal typeof GABA transporter, also
inhibited GABA uptake. Creatine, taurine,glycine, and betaine have, if any, a
small inhibitory effecton GABA uptake.
Effect of Na+ and Cl- Chemical Gradient and
Electrical Membrane Potential on Creatine Uptake in BBMV
The effects of either electrical membrane potential, Na+, or
Cl-chemical gradients on [14C]-creatine uptake in renal
cortexBBMV are shown in Figure
3. Electrical membrane potential wascreated by an outwardly
directed K+ gradient in the presenceof valinomycin. When required,
the voltage across the membranewas clamped at 0 by equal internal and
external K+ concentrationsin the presence of valinomycin. An
inside directed NaCl chemicalgradient was created by the addition of 250 mM
NaCl to the extravesicularbuffer, given that the intravesicular buffer is
nominally freeof NaCl. The Na+, Cl-, or NaCl chemical
gradient was abolishedby isosmotic replacement of NaCl with either choline
Cl, Nagluconate, or K gluconate, respectively.
Figure 3. A 15-s [14C]-creatine uptake into BBMV in the presence and
absence of electrical membrane potential and/or either Na+ or
Cl- chemical gradient. The uptake buffer contained (in mM) 50
HEPES-Tris (pH 7.5); 0.020 valinomycin; and 200 mannitol, 250 NaCl (electrical
and NaCl gradient), or 100 K gluconate and 250 NaCl (only chemical NaCl
gradient) or 100 K gluconate and 250 Na gluconate (only chemical
Na+ gradient) or 100 K gluconate and 250 N-methyl-glucamine-Cl
(only Cl- gradient) or 100 K gluconate and 500 mannitol (without
gradients) or 700 mannitol (only electrical gradient). The concentration of
[14C]-creatine in the uptake buffer was 5 µM (n = 3).
*, P < 0.001, compared with electrochemical NaCl
gradient conditions.
The results revealed that clamping the membrane voltage at 0inhibited
creatine uptake, and this inhibition was increasedby either Na+ -
or Cl--free conditions. The transport of creatinerequires the
presence of both Na+ and Cl- in the extravesicular
buffer,because the inhibition induced by the absence of only one ofthe two
ions was not different from that seen in the absenceof NaCl.
Kinetic Study of Creatine Uptake
The relationship between creatine uptake in BBMV and substrate
concentrationwas obtained by measurement of the initial rate of uptake (15-s
incubation)at varying concentrations of creatine over a range of 1 to 300
µM.The initial uptake rate saturates with increasing concentrationsof
creatine and the hyperbolic shape of the curve
(Figure 4)suggested the
participation of a single transport system inthe uptake process. This
conclusion was supported by the linearityof the Eadie-Hofstee plot. The
calculated apparent Km and Vmaxfor
creatine uptake are 15.3 ± 3 µM and 476 ±6.5 pmol/mg protein
per 15 s, respectively.
Figure 4. Initial rate (15-s) of creatine uptake versus increasing
concentrations of extravesicular creatine. The composition of the intra- and
extravesicular buffers is described in the Materials and Methods section. The
external creatine concentration ranged from 1 to 300 µM. (Inset)
Eadie-Hofstee plot of data (n = 5).
Na+:Cl-:Creatine Stoichiometry
Because an inside-negative membrane potential stimulated theNaCl-dependent
[14C]-creatine uptake, which suggests the nettransfer of positive
charge across the membrane during the uptakeprocess, we evaluated the
stoichiometry among Na+, Cl-, andcreatine using the
activation method. We measured the dependenceof the initial rate of creatine
uptake (15-s incubation) onthe extravesicular concentration of either
Na+ (keeping theextravesicular concentration of Cl-
constant at 250 mM) or Cl-(keeping the extravesicular
concentration of Na+ constant at250 mM). In both cases, the
intravesicular buffer was nominallyfree of NaCl, and the membrane vesicles
were voltage clamped(equal internal and external K+ concentration
and valinomycin)to avoid the effects of a membrane voltage on the uptake
rate.
Figure 5 reveals that the
initial rate of creatine uptake increasedwith increasing Na+
concentration. The sigmoidal shape of thecurve indicates that the creatine
uptake system interacts withmore than one Na+ ion. The number of
Na+ ions interacting withthe carrier was determined by fitting the
data to a Hill-typeequation:
(1)
and plots of V versus V/[Na+]nwere
constructed for a Na+ concentration range of 10 to 250mM. When
n = 1.7, the plot was close to a straight line. Theseresults suggest
that at least two Na+ ions are involved pertransport of one
creatine molecule. The approximate value ofK0.5 for
Na+ (i.e., the concentration of Na+ necessary
for half-maximalactivation) was 183 mM.
Figure 5. Dependence of creatine uptake on Na+ concentration. The uptake
buffer contained (in mM) 50 mM HEPES-Tris (pH 7.5), 100 K gluconate, and
different concentrations of Na+, which were made by substitution of
NaCl with NMGCl. Membrane vesicles were voltage clamped with 20 µM
valinomycin. Uptake of 5 µM creatine was measured for 15 s (n =
3). Na+-dependent creatine uptake versus Na+
concentration. (Inset) Hill-type plot of the data, in which the initial
velocity (V, in pmol creatine/mg protein 15 s) was plotted against
V/[Na+](1.7), r = 0.975.
Figure 6 shows that creatine
uptake increased with increasingconcentrations of Cl- in an
hyperbolic manner. The plot of Vversus
V/[Cl-]n was a straight line when
n = 1, which suggeststhe involvement of one Cl- per
transport of one creatine molecule.The approximate value of
K0.5 for Cl- was 57 mM.
Figure 6. Dependence of creatine uptake on Cl- concentration. The
different concentrations of Cl- were made by substitution of NaCl
with Na gluconate. Other details as in
Figure 5.
Cl--dependent creatine uptake versus Cl-
concentration. (Inset) Hill-type plot of the data, in which the initial
velocity (V, in pmol creatine/mg protein 15 s) was plotted against
V/[Cl-]1, r = 0.991.
Homeostatic control of serum creatine concentration might operateat
several sites: (1) intestinal absorption, (2) biosynthesis
ofcreatine, (3) uptake and retention of creatine by muscle andnerve
cells, and (4) kidney reabsorption of creatine. Althoughcreatine
transport has been investigated in several cell types
(13,14,15,16,17,18,23,24),
thecharacterization of the mechanism(s) involved in kidney creatine
reabsorptionhas not been addressed.
In the present study, we characterized creatine transport inBBMV isolated
from rat renal cortex. The conclusions of thisstudy are that (1) the
vesicles transiently accumulate creatinein the presence of an inwardly
directed NaCl gradient; (2) theuptake of creatine is Na+-
and Cl--dependent and electrogenic,with a
Na+:Cl-:creatine stoichiometry of 2:1:1, which results
inthe transfer of a net positive charge across the membrane; and(3)
the uptake system has a high affinity for creatine (15 µM).
Previous studies carried out in different cell types
(13,14,15,16,17,18,23,24)
didnot evaluate the role of Cl- on creatine active transport and
showedonly the Na+ dependency of the transport system. Studies
carriedout with COS-7 cells
(5), transfected with the cDNA
encodinga rabbit brain CREAT, and in Xenopus laevis oocytes that
expresshuman heart CREAT
(25), revealed that creatine
transport isNa+- and Cl--dependent, and in the latter
expression systemit was found that at least two Na+ and one
Cl- were requiredto transport one creatine molecule.
The Kµ value for creatine transport obtained in the
currentstudy (15 µM) is 10 times lower than the value reported
(26)for rat creatine plasma
concentration (135 µM). The Kµvalue is similar to
that found in COS-7 cells transfected withhuman brain CREAT
(6), red blood cells
(23), Xenopus oocytes
expressinghuman heart CREAT
(25), and monocytes and
macrophages (16);is lower
than that obtained in astroglia cell cultures
(17),red blood cells
(24), L6 myoblasts
(27), and in HEK-293
(7)and COS-7-cells
(5) that express rabbit
brain/spinal cord CREAT;and is an order of magnitude lower than that measured
in a myoblastcell line (15)
and in HEK-293 cells transfected with cDNA encodingbovine CREAT
(28).
As reported for other cell types
(16,17,23)
and for the clonedCREAT cDNA expressed in COS-7 cells
(6), Xenopus oocytes
(25),and HEK-293 cells
(7), GPA strongly inhibited
radioactive creatineuptake in renal BBMV, as it did a 100-fold excess of
unlabeledcreatine. GABA, ß-alanine, and betaine inhibited creatine
uptakeby 15%, and glycine, carnitine, taurine, and choline did not
significantlyaffect the creatine uptake.
The human creatine transporter is strongly related to a subfamilyof
protein sequences that includes the transporters of taurine,betaine, and
GABA. The results presented in this study stronglysupport the idea that renal
cortex BBMV have a specific CREAT-preferringtransport system different from
GABA, betaine, or taurine transporters.Thus, betaine and taurine have a small
effect on creatine transport.Furthermore, the substrate specificity of GABA
transport andthat of creatine transport in renal cortex BBMV were quite
different(see Table 1).
Creatine uptake was abolished by a 100-fold excessof unlabeled creatine,
whereas GABA uptake was strongly inhibitedby a 100-fold excess unlabeled GABA
but was minimally inhibitedby creatine. It is interesting that GPA, a
high-affinity alternativesubstrate for creatine transport in several tissues
(16,17,23),
alsostrongly inhibits GABA uptake (80% inhibition). Similarly, nipecotic
acid,described as an inhibitor of the neuronal type of GABA transporter
(29),inhibited creatine and
GABA uptake by the same magnitude.
In conclusion, to our knowledge this is the first report showingthat the
apical membrane of the cortical tubule of rat kidneyhas a high-affinity, 2
Na+/Cl-/creatine transport system. Becausethe
NaCl-independent creatine uptake in BBMV is low, the characterized2
Na+/Cl-/creatine transport system must be the one
mediatingthe first step of kidney creatine reabsorption.
Acknowledgments
This work was supported by a grant from the Spanish Ministeriode Ciencia y
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Received for publication November 29, 2000.
Accepted for publication February 19, 2001.
This article has been cited by other articles:
E. Boehm, S. Chan, M. Monfared, T. Wallimann, K. Clarke, and S. Neubauer Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine
Am J Physiol Endocrinol Metab,
February 1, 2003;
284(2):
E399 - E406.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-aminobutyric acid (GABA) = ß-alanine = betaine, whereas
carnitine, glycine, taurine, and choline were without effect. This pattern of
inhibition differs from that observed for GABA uptake (unlabeled GABA = GPA
> ß-alanine > nipecotic acid >> creatine). The conclusion drawn
was that the apical membrane of the renal cortical tubules contains an active,
high-affinity, electrogenic, 2 Na+/1 Cl-/creatine
cotransporter. 
; NaCl isosmotically replaced by K gluconate)
and presence ([UNK]) of inwardly directed NaCl gradient as a function of time.
Other details as in 
