THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
c)1991 by
Vol. 266, No. 15, Issue of May 25, pp. 9392-9396, 1991
Printed in U.S.A .
The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Acute Alterations in Acid-Base Balance
on Rat Renal
Glutaminase and Phosphoenolpyruvate Carboxykinase Gene
Expression*
(Received for publication, October 22, 1990)
Jung-Joo Hwang and NormanP. CurthoysS
From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523
During chronic acidosis, the levels of the rat renal
mRNAs that encode the mitochondrial glutaminase
(GA) and cytosolic phosphoenolpyruvate carboxykinase (PCK) are increased 6-fold. Following acute recovery of chronic acidosis, the levels of the two mRNAs
are rapidly and coordinately decreased, returning to
normal within 13-17 h. In contrast, the increases in
GA and PCK mRNAs during acute onset of acidosis
occur with very differentkinetics. The increase in
PCKmRNA occurs rapidly and reaches a maximum
within 7 h, whereas the GA mRNA is increased after a
4-7-h lag and then plateaus at 14-17 h. Treatment
with dexamethasone or with cAMP analogs significantly increases the level of renal PCK mRNA but has
no effect on the level of GA mRNA. Nuclear run-on
experiments indicate that the acute induction of PCK
mRNA is primarily due to an increased rate of transcription. However, transcription of GA mRNA is unaffected by acute acidosis. Therefore, the changes in
the two mRNAs are temporally coordinated but occur
through different mechanisms. Furthermore, the inductive effects of acidosis are not mediated solely
through glucocorticoid or cAMP regulatory elements.
olpyruvate carboxykinase (12) which are the key regulatory
enzymes of renal ammoniagenesis and gluconeogenesis, respectively. Thethree enzymes aredistributed differently
along thevarious segments of the normal rat nephron.However, in response to metabolic acidosis, the three activities are
increased solely in the SI and S p segments of the proximal
convoluted tubule (10,13,14). Studies using dissected tubules
have established that increased glutamine
metabolism occurs
solely within the same segmentsof the nephron (15).
Previous studies have established that increased glutaminase (16, 17) and phosphoenolpyruvatecarboxykinase (12, 18)
activities are due to increased rates
of synthesis that correlate
with increasedlevels of their respective mRNAs. Induction of
carboxykinase activity occurs rapidly and is complete within
14-20 h. In contrast,full induction of the glutaminaserequires
5-7 days. However, the difference in half-lives of the two
proteins is sufficient to account for the difference in time
required to reacha new steady-state level (12, 16).Therefore,
the adaptation in the two enzymes could be initiated by a
common stimulus andcould share a common mechanism.
In order to further characterize the mechanism by which
altered acid-base balance leads cell-specific
to
changes in renal
gene expression, we have characterized the kinetics of the
adaptations in GA’ and PCK mRNA levels and transcription
rates thatoccur in response to alterations in acid-base balance
Increased renal ammoniagenesis andgluconeogenesis from and tovarious stimuli. The results of this study indicate that
glutamine are essential in order to partially restore acid-basethe coordinate adaptations in the two mRNAs are not mebalance during metabolic acidosis (1, 2). Increased renal ex- diated solely through glucocorticoid or cAMP regulatory eletraction and metabolism of plasma glutamine during acute
ments and may proceed through different mechanisms.
acidosis is accomplished by an increased availability of substrate and the rapidremoval of the productsof the glutamate
MATERIALS ANDMETHODS
dehydrogenase reaction. Plasma glutamine is increased as a
[a-”PIdCTP (specific activity, 3000 Ci/mmol), [a-’”P]CTP (speresult of increased synthesis in muscle and liver (3, 4). The
activity, 3000 Ci/mmol), [‘HH]UTP (specificactivity, 46.8 Ci/
initial increase in renal extraction correlates
with the increase cific
mmol), Genescreen, and GeneScreen Plus were obtained from Du
in plasma glutamineconcentration (5). Increased fluxes Pont-New England Nuclear. RNase T1 and bovine serum albumin
through renal mitochondrial glutaminase and glutamate de- were purchased from GIBCO/BRL. Nucleoside-5’-diphosphatekihydrogenase are also favored by a prompt acidification of the nase, yeast tRNA, glutamatedehydrogenase, restriction enzymes, and
were products from Boehringer Mannurine that promotes the removal of intracellular ammonium 8-(4-~hlorophenylthio)-cAMP
ions (6) and by activation of a-ketoglutarate dehydrogenase heim GmbH. Dexamethasone was purchased from Elkins-Sinn, Inc.
The oligolaheling kit andACS solution were obtained from Pharmacia
due to a decreased intracellular pH (6-8).
LKB Biotechnology Inc. and Amersham Corp., respectively. Sucrose
Increased renal metabolism of glutamine is sustained duringwas purchased from Schwarz/Mann Biotech. RNasin and Ti. and T:,
chronic acidosis even though all of the initial adaptations are RNA polymerases were purchased from Promega and U. S. Biochempartially compensated (9). In the rat, this is
accomplished by ical Corp., respectively. All other biochemicals were purchased from
(10) and either Sigma or Fisher. The pPCK-10, which encodes rat phosphoentheinduction of themitochondrialglutaminase
glutamate dehydrogenase (11) and the cytosolic phosphoen- olpyruvate carhoxykinase, was obtained from R. Hanson (Case West-
* This work was supported in partby National Institutesof Health
Grant DK-37124. The costs of publication of this articlewere defrayed
in part by the payment of page charges. This article must therefore
he hereby marked “advertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate thisfact.
$ T o whom correspondence should he addressed.Tel: 303-4915566.
ern Reserve University School of Medicine) (19).ThepHF@A-1,
which encodes human @-actin,
was obtained from L. Kedes (Stanford
University School of Medicine) (20).
Animals-Male Sprague-Dawley rats weighing 200-250 g were
The abbreviations used are: GA, glutaminase; PCK, phosphoenolpyruvate carboxykinase; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
9392
ofEffect AcidosisExpression
onGene
Renal
purchased from Zivic-Miller Laboratories and maintained on Purina
rat chow. Chronic metabolic acidosis was induced by providing 1.5%
NH,CI as thesole source of drinking water.After 5 days, aset of rats
was stomach-loaded with20 mmol of NaHCO,%/kgof body weight to
acutely correct theacidosis. Acute metabolic acidosis was induced by
stomach loading rats with 20 mmol of NH,CI/kg of body weight and
then allowing them to drink 1.5% NH,CI. All other effectors were
injected intraperitoneally.
cDNA and cRNA Probes-The original pGA-1 and pGA-2 cDNAs
(21) were used to screen X g t l O cDNA libraries that were synthesized
by oligo(dT) priming and random priming
of rat kidney and rat brain
mRNAs, respectively. The rat kidney library yielded XGA-13 (2.1kilobase insert), while the brain libraryyielded XGA-104 (2.7-kilobase
insert). Both inserts were subcloned into pBluescript II-SK(-), and
the resulting plasmids contained all of pGA-1 and pGA-2. The Sac1
fragment of pGA-13, BglII fragment of pPCK-10, and BarnHI fragment of pHFpA-1 were isolated (22, 23), labeled by random priming
(24,25), andused as probesfor Northern analysis. The isolated cDNA
fragments derived from pPCK-10 and pHF@A-1were also subcloned
into pBIuescript II-SK(-) and along with linearized pGA-104 were
used to synthesize cRNA probes. Sense and anti-sense RNAs were
transcribed using a modification of the proceduredescribed by Krieg
and Melton (26). The reaction mixture contained 2 mM spermidine,
500 units/ml RNasin, 10 mM NaCI, 10 mM dithiothreitol, 80 pg/ml
bovine serum albumin,22 mM MgCI?, 4 mM each of ATP, CTP, GTP,
and UTP, 0.1% Triton X-100, 40 mM Tris-HC1, pH 7.5, and 1-2 pg/
50 p1 linearized plasmid. The reaction was initiated by adding 10
units of T:,or T; RNA polymerase and incubateda t 30 "C for 90min.
The reaction was stopped by adding 1 pg of RNase-free DNase I. The
reaction mixture was centrifuged through a SephadexG-50spin
column. The eluant was extracted with phenol:CHCl:,, precipitated
with ethanol, and stored a t -70 "C until used.
Northern Ana/ysis-TotalRNA
was isolated from rat kidneys
according to theprocedure of Chirgwin et al. (27). Aliquots containing
15 pg of the isolated RNAs were fractionated by electrophoresis ona
1% agarose gel containing 3% formaldehyde (22). Fractionated RNAs
were transferred to GeneScreen and hybridized (28) with the DNA
probes that were labeled (2 X loGcpm/ml) by random priming (25).
The hybridized filters were exposed to Kodak X-Omat film. Radioactivity from GeneScreenwas determined by liquid scintillation
counting andcorrected for backgroundby subtraction of radioactivity
found on blank squarescut from thesame filter. All data were
calculated as specific hybridization relative to thatof @-actin.
Nuclear Run-on Transcription Assay-Nuclei wereisolated and
transcription run-on experiments were performed as described (2931). This methodyielded approximately 0.5 X 10' nuclei/g of kidney,
wet weight. The transcription reaction mixture (200 pl) contained
0.21 p M [w'"P]CTP (3000 Ci/mmol), 25% glycerol, 75 mM sodium
Hepes (pH7.5), 100 mM KCI, 5 mM magnesium acetate, 1mM MnCI?,
50 p~ EDTA, 1 mM ATP, 0.5 mM G T P and UTP,4 mM dithiothreitol,
0.1 mg/ml heparin, 8.8 mM phosphocreatine, 40 pg/ml creatine phosphokinase, 200 units/mlRNasin, 0.1 mg/ml nucleoside-5'-diphosphate kinase, andapproximately 2 X 10' nuclei. After 10 min of
incubation at 25 "C, nuclei were digested with ribonuclease-free
DNase I. Yeast tRNA was added, and nuclear RNA was isolated by
adding a solution containing 4 M guanidinium thiocyanate, 25 mM
sodium citrate(pH 7.0), 0.5% sodiumlauroylsarcosine,
0.1 M 2mercaptoethanol, 2 M sodium acetate(pH 4.0), water-saturated
phenol, and ch1oroform:isoamyl alcohol (24:l). The labeled nuclear
RNA was precipitated with isopropyl alcohol, washedin 70% ethanol,
dissolved, and centrifuged through a Sephadex G-50 spincolumn. In
some reactions, cu-amanitin (2 pg/ml)wasadded
to inhibitRNA
polymerase 11-directed transcription (32). In a typical reaction, 2-5
X 10' cpm were incorporated intoRNA. Aliquots containing 10 pg of
the anti-sense RNAs were immobilized on a GeneScreen Plus mem& Schuell"Minifold 11" apparatus (33).
brane usingaSchleicher
Hybridizations were carried out in 50% formamide, 0.25 M sodium
phosphate (pH 7.2), 0.25 M NaCI, 1 mM EDTA, 100 pg/ml denatured
salmon sperm DNA,10 pg/ml poly(A') RNA, 7% sodium dodecyl
sulfate and 10' cpm/ml RNAs a t 53 "Cfor 3 days. After treating with
RNase and washing, the hybridized nuclear RNAs were quantitated
by autoradiography and densitometry using a computerized Microscan 2000 image analyzer (Technology Resources, Inc., Nashville T N )
or by liquid scintillation countingof s1ot.sexcised from the hybridized
filter.Hybridization efficiency (-30%) was determined simultaneously by quantitating the extentof binding of "H-labeled sense PCK
HNA.All data were calculated as specific hybridization relative to
that of &actin.
9393
RESULTS
Acute Recovery from Chronic Acidosis-The development
of chronic acidosis caused a 6-fold increase in the levels of
GA and PCK mRNA(Fig. 1).In contrast, thelevel of P-actin
mRNA was unaffected by changes in acid-base balance. Recovery from chronic acidosis causes a rapid decrease in PCK
activity(34)andinthe
relative level of translatable GA
mRNA (17). Therefore,the effect of acute recovery from
chronic acidosis on the levels of GA and PCK mRNAs was
determined. Total RNAwas isolated from 5-day acidotic rats
a t various times after treating with
NaHCOs. The RNAswere
subjected to Northern blot analysis and probed with one of
the two specific cDNAs. Each blot was rehybridized with the
@-actinprobe, and the ratiosof the GA and PCK mRNAs to
that of P-actin mRNA were determined. The decrease in the
relative levels of the two mRNAs was initiated within 1 h
after acute recovery and returned to normal within 13-17 h
(Fig. 2). In each case, the decrease occurred with first order
kinetics and an apparent half-life of5-6 h. The observed
N
A
N
A
LL
GAPCK
-
PAC
-
- 28s
ai-18s
FIG. 1. Effect of onset of acidosis on the relative abundance
of renal 8-actin( ~ A c ) GA,
,
and PCK mRNAs. Aliquots containing 15 pg of total cellular RNA purified from the kidneys of normal
rats ( N ) or rats that were made chronically acidotic ( A ) were characterized by Northern analysis.
I
I
I
4
I
I
I
I
I
I
8
12
16
HOURSOFRECOVERY
I
I
20
I
I
I
24
FIG. 2. Effect of acute recovery from chronic acidosis on
the relativelevels of GA and PCK mRNAs. The decrease in GA
(A-A) levels was measured at
mRNA (A-A) andPCKmRNA
various times after treating 5-day chronic acidotic rats with 20 mmol
of NaHCOZl/kgof body weight. Total RNAswere subjected to Northern analysis. All data were calculated as specific hyhridization relative
to that of @-actin.Eachpointisthe
mean +. S.D. of the values
obtained from three rats. Therelative levels of mRNA for individual
time points ( R ) were replotted in the inset,where R,, and RS are
equal to the relative steady state levels for the mRNA under acidotic
and normal conditions, respectively.
Effect of Acidosis o n Renal Gene Expression
9394
results suggest that the decrease in GA and PCK mRNA
abundance during acute rccovery may be initiated by a common effector and may occurthrough a coordinate mechanism.
Acute Onset of Acidosis-The level of PCK mRNA is increased rapidly following acute onset of acidosis (35). T o
determine if GA and PCK mRNAs are coordinatelyinduced,
the increases in the relative levels of the two mRNAs were
determined a t various times after treating rats with NH&l
(Fig. 3). The increase in PCK mRNA was initiated within 1
h after acute onsetof acidosis and reached a plateau after 7 h
at a level that was 6-fold greater than normal. In contrast,
the level of GA mRNA was increased following a 4-7 h lag
and then plateaued at a level that was 8-fold greater than
normal. Thedifference in kineticssuggests that theinduction
of PCK and GA mRNAs may occur through different mechanisms or inresponse to different effectors.
Effect of Acidosis on Transcription-To identify the
possible
mechanism responsible for the elevatedlevel of GA and PCK
mRNAs, transcription run-on assays were performed using
rat renal nuclei isolated at different times following acute
onset of acidosis (Fig. 4). The rate of [a-"'Pp]CTP incorporationintototalnuclearRNA
was linearfor 60 min.The
observed hybridizations were inhibited by greater than 90%
by inclusion of 2 pg/ml a-amanitin. Thus, thelabeled RNAs
that hybridize were specific products of RNA polymerase I1
transcription. The rate of P-actin mRNA transcription was
unaffected by acidosis. The relative transcription rate of the
PCK gene increased %fold within 2 h after onset of acute
acidosis, reached a maximum of 4-fold induction by 6 h, and
then decreased slightly by 20 h (Fig. 5). The initial increase
in transcription exceeded the initial increase in PCK mRNA.
Therefore, enhancementof transcription can accountfor the
initial inductionof PCK mRNA. However, increased stability
of PCK mRNA may contribute to maintenanceof the maximally induced steady state level. In contrast, the rate of GA
transcription was unaffected by onset of acute acidosis (Fig.
4).
Effect of Dexamethasone and CAMP-The abundance of
renal PCK mRNA is alsoincreased by treatment with glucocorticoids or with various derivatives of CAMP. The effect of
glucocorticoids on renal PCK mRNA
alsooccurs through
transcriptional regulation (36). Therefore, the responses of
the two mRNAs to pretreatment with the potential
effectors
were determined. Dexamethasone and the chlorophenylthioand dibutyryl-derivatives of cAMP caused a significant increase in renal PCK mRNAs(Fig. 6). However, neither treatment affected the level ofGA mRNA. Thus, the effect of
acidosis isnotmediated
solely through glucocorticoid or
cAMP regulatory elements.
DISCUSSION
I
N
I
4
I
I
a
I
I
I
I
I
16
HOURS OF ACUTE ACIDOSIS
12
I
20
I
I
24
FIG.3. Effect of acute onset of acidosis on the relative levels
of GA and PCK mRNAs. The increase in GA mRNA (A-A) and
PCK mRNA (A-A) was measured at various times following onset
of acute acidosis induced by stomach loading rats with 20 mmol of
NH,Cl/kg of body weight. Total RNAs from the kidneys were subjectedto Northern analysis. All data were calculated as specific
hybridization relative to that of @-actin.Each point is the mean f
S.D. of the values obtained from at least three rats.
N
6h
-
20h
-
The onsetof metabolic acidosis initiates a well coordinated
series of adaptations in the interorgan metabolism of glutamine. In normalacid-base balance, the splanchniccatabolism
of amino acids generates nearly equivalent amounts
of bicarbonate and ammonium ions that are used to generate urea
(37). Acidosis decreases urea synthesis and increases hepatic
synthesis of glutamine (38). This adaptation is balanced by
an increased renal catabolism of glutamine that serves two
functions. Excretion of the resulting ammonium ions in the
urine provides a n expendablecation which facilitatesthe
excretion of acids while conserving sodium
and potassium
ions. In addition, the subsequent conversion of the carbon
20h
+
BAc -
B-
GA -
PCK
FIG.4. Nuclear run-on transcription assay using isolated
renal nuclei. Renal nuclei from normal ( N )and acute acidotic rats
( 6 h and 20 h ) were incubated with [n-:"P]CTP in the presence (+)
or absence (-) of 2 pg/ml n-amanitin. The[R'P]CTP-labelednuclear
RNA was hybridized toa membrane containing 10 pg of @-actin
( ~ A C GA,
) , and PCK anti-sense RNAs. Theblank lane ( B )contained
no RNA.
N
4
8
12
16
20
24
HOURS OFACUTE ACIDOSIS
FIG. 5. Comparison of the rates of PCK gene transcription
and the level of total PCK mRNA. The relative rates of transcription of the PCK gene (hatched bar) and the relative levels of PCK
mRNA (A-A) were determined using rat renal nuclei and total RNA
isolated from normal rats ( N )and from rats at various times following
onset of acute acidosis. All data were calculated as specific hybridization relative to that of @-actin.Each point is the mean f S.D. of
the values obtained from at least three rats.
Effect of Acidosis
on
Renal Gene Expression
0GA mRNA
PCK mRNA
i
rJY
2
1
Control
Dex
3x-24h
4h
Dex
CPcAMP
Glc+
3x-24h
Bt2cAMP
lh
FIG.6. Effect of glucocorticoid and cyclic adenine nucleotides on the renal levels of GA and PCK mRNAs. Total RNA
was isolated from rats that were injected with saline (Control), 5 mg
of dexamethasone (Dex)/kg of body weight, 25 mg of 8-(4-chlorophenylthio)-CAMP (CPcAMP)/kg of body weight, or 25 mg of dibutyryl cAMP (BtcAMP)/kgof body weight. 2 h after stomach loading
with 5 g of glucose/kg of body weight Northern analysis was performed using total RNA. All data were calculated as specific hybridization relative to that of @-actin. Each point is the mean? S.D. of
the values obtained from a t least three rats.
9395
hepatic PCK mRNA. In addition, the 3"untranslated region
of PCK mRNA containsAU-rich regions (42). Similar motifs
participate in theselective degradation of a number of transiently expressed mRNAs (43-45). A single renal pH-responsive signal transfer mechanismcould initiate both processes.
The kineticsof induction of PCK andGA mRNAs following
acute onset of acidosis differ significantly. The increase in
PCK mRNA begins immediately and is nearly complete before the pronounced increase in GA mRNA is initiated. The
observed difference suggeststhat thetwo responses may result
from different processes. This hypothesis was confirmed by
the observation that in contrast to PCK mRNA, transcription
of GA mRNA is not increased during acute
acidosis. The rate
of renal GA transcription is significantly lower than that of
PCK mRNA. As a result,the level of radioactivity that
hybridized to theGA antisense RNAwas too low to accurately
quantitate by directscintillationcounting.
However, the
bands were easily quantitated by densitometry. When the
two
methods are used to calculate the
increase in PCK transcription, they yield identical data. In addition, the synthesis of
transcripts which hybridize to the P-actin,
GA, and PCK antisense RNAs were inhibited greater than90% by the addition
of a level of a-amanitin that specifically blocks RNA polymerase I1 activity. These observations support the conclusion
that transcription of the GA gene is not increased during
metabolicacidosis. The 3"untranslated region of the GA
mRNA also contains multiple AU-rich regions' which might
participate in a pH-responsive alteration in GA mRNA stability.
Acute recovery from chronic acidosis causes a coordinate
decrease in the levels of PCK and GA mRNAs. Blood pH is
only slightly decreasedcompared with normal ina chronically
acidotic rat (9). Thus, it is likely that the mechanism responsible for the initial induction of PCK mRNA during acute
acidosis may differ from that which sustains the steady state
level observed during chronic acidosis. The increase in PCK
transcription during chronic acidosis accounts for only 15%
of the fully induced level of PCK mRNA.3 This slight increase
in PCK transcription is rapidly reversed during acute recovery. Therefore, recovery from chronic acidosis may alsoresult
as
from coordinate changes in the stabilityof the PCK andGA
mRNAs. The data presented in the
accompanying paper (46)
demonstrate that the increases in PCK and
GA mRNA levels
whichoccurin
LLC-PK-F' cells in response to decreased
medium pH and bicarbonate concentration are due, at least
in part, to their increased stability.
skeleton of glutamine to glucose generates bicarbonate ions
that are added to the plasma and partially compensate the
systemic acidosis. Increased rat renal ammoniagenesis and
gluconeogenesis are sustainedby the cell-specific induction of
the mitochondrial glutaminase(10) and phosphoenolpyruvate
carboxykinase (13). Given their coordinate function, it was
anticipated that the inductionof the two genes would utilize
a commonmechanism. However, the results of this study
suggest that the adaptation in thelevels of the two mRNAs
is temporally coordinated butoccurs by different mechanisms.
The acute increase in PCK mRNA that occurs following
onset of acidosis apparently resultsfrom an increased rateof
transcription. The initial increase in the transcription rate
measured by the nuclear run-on assays can account for the
initial increase inPCKmRNA
(Fig. 5). The increasesin
hepatic PCK mRNA thatoccur in response toglucocorticoids
or aglucagon-mediated increase in cAMP result from increased transcription (39, 40). Similarly, glucocorticoids increase transcription of the renal PCKgene (36). However, the
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