1 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870; and 2 Department of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria
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ABSTRACT |
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Increased renal catabolism of plasma glutamine during metabolic
acidosis generates two ammonium ions that are predominantly excreted in
the urine. They function as expendable cations that facilitate the
excretion of acids. Further catabolism of -ketoglutarate yields two
bicarbonate ions that are transported into the venous blood to
partially compensate for the acidosis. In rat kidney, this adaptation
is sustained, in part, by the induction of multiple enzymes and various
transport systems. The pH-responsive increases in glutaminase (GA) and
phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are
reproduced in LLC-PK1-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA
mRNA. The 3'-untranslated region of the GA mRNA contains a direct
repeat of an eight-base AU sequence that functions as a pH-response
element. This sequence binds
-crystallin/NADPH:quinone reductase
with high affinity and specificity. Increased binding of this protein
during acidosis may initiate the pH-responsive stabilization of the GA
mRNA. In contrast, induction of PEPCK occurs at the transcriptional
level. In LLC-PK1-FBPase+ kidney cells, a
decrease in intracellular pH leads to activation of the p38
stress-activated protein kinase and subsequent phosphorylation of
transcription factor ATF-2. This transcription factor binds to
cAMP-response element 1 within the PEPCK promoter and may enhance its
transcription during metabolic acidosis.
glutamine metabolism; glutaminase; phosphoenolpyruvate carboxykinase; LLC-PK1-fructose 1,6-bisphosphatase cells
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RENAL GLUTAMINE METABOLISM |
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GLUTAMINE IS AN IMPORTANT metabolic fuel that is constitutively metabolized in liver (34), intestinal epithelium (55), lymphocytes (59), brain (48), and various transformed cells (9). In contrast, renal catabolism of glutamine is activated only during metabolic acidosis (77). During normal acid-base balance, the kidney extracts and metabolizes very little of plasma glutamine (75). The measured renal arteriovenous difference for plasma glutamine is normally <3% of arterial concentration. However, ~20% of plasma glutamine is filtered by the glomeruli and enters the lumen of the nephron. The filtered glutamine is reabsorbed primarily by the epithelial cells of the proximal convoluted tubule (72). It is initially transported across the apical brush-border membrane, and subsequently most of the recovered glutamine is returned to the blood via transport across the basolateral membrane. The specific transporters that are responsible for the transcellular flux of glutamine have not been identified.
Utilization of the small fraction of extracted plasma glutamine requires its transport into the mitochondrial matrix, where glutamine is deamidated by glutaminase (GA) and then oxidatively deaminated by glutamate dehydrogenase (GDH). Glutamine uptake occurs via a mersalyl-sensitive electroneutral uniporter (68). The mitochondrial glutamine transporter was recently purified from rat kidney and shown by reconstitution in lipid vesicles to be specific for glutamine and asparagine and inhibited by various thiol reagents (43). Kinetic measurements indicated that the rate of glutamine transport in isolated rat renal mitochondria is not rate limiting for glutamine catabolism (19, 47). However, the activity of either the mitochondrial glutamine transporter or GA must be largely inactivated in vivo during normal acid-base balance to account for the effective reabsorption of glutamine. During normal acid-base balance, approximately two-thirds of the ammonium ions produced from glutamine are trapped in the tubular lumen and are excreted in a slightly acidified urine (74).
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ACUTE ACIDOSIS |
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The maintenance of blood acid-base balance is essential for
survival. Increased renal ammoniagenesis and gluconeogenesis from plasma glutamine constitute an adaptive response that partially restores acid-base balance during metabolic acidosis (3).
Thus the renal catabolism of glutamine is rapidly activated after the acute onset of metabolic acidosis. Within 1-3 h, the arterial plasma glutamine concentration is increased twofold (39),
due primarily to an increased release of glutamine from muscle tissue (70). Significant renal extraction of glutamine becomes
evident as the arterial plasma concentration is increased. Net
extraction reaches 30% of the plasma glutamine, a level that exceeds
the percentage filtered by the glomeruli. Thus the direction of the basolateral glutamine transport must be reversed in order for the
proximal convoluted tubule cells to extract glutamine from both the
glomerular filtrate and the venous blood. In addition, the transport of
glutamine into the mitochondria may be acutely activated
(69). Further responses include a prompt acidification of
the urine that results from an acute activation of the apical Na+/H+ exchanger (38). This
process facilitates the rapid removal of cellular ammonium ions
(78) and ensures that the bulk of the ammonium ions
generated from the amide and amine nitrogen of glutamine are excreted
in the urine. Finally, a pH-induced activation of -ketoglutarate
dehydrogenase reduces the intracellular concentrations of
-ketoglutarate and glutamate (54). Thus increased catabolism initially results from a rapid activation of key transport processes, an increased availability of glutamine, and a decreased concentration of the products of the GA and GDH reactions.
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CHRONIC ACIDOSIS |
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During chronic metabolic acidosis, many of the acute adaptations
are partially compensated for and the arterial plasma glutamine concentration is decreased to 70% of normal (3). However,
the kidney continues to extract more than one-third of the total plasma glutamine (75) in a single pass through the organ (Fig.
1). Renal catabolism of glutamine is now
sustained by increased expression of the genes that encode
mitochondrial GA (10), mitochondrial GDH
(83), and cytoplasmic phosphoenolpyruvate
carboxykinase (PEPCK) (4). The tubular distributions of
PEPCK, fructose 1,6-bisphosphatase (FBPase), and glucose 6-phosphatase
were determined in microdissected segments of the rat nephron
(30). All three activities, and thus the pathway of
gluconeogenesis, are expressed solely within the proximal tubule.
However, after the onset of acidosis, the rapid increase in PEPCK
occurs only within the S1 and S2 segments of the proximal tubule
(4). The more gradual increase in the levels of the
mitochondrial GA (10, 84) and GDH (83) also occurs solely within the proximal convoluted tubule. Previous micropuncture studies (67) and assays using microdissected
nephron segments (22) have also established that the
preponderance of renal ammoniagenesis in normal or acidotic rats occurs
within the convoluted portion of the proximal tubule. The decreases in plasma pH and HCO
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The increase in apical Na+/H+ exchanger
activity sustains the acidification of the fluid in the tubular lumen
and ensures the urinary excretion of the ammonium ions. Thus the
increased renal ammoniagenesis continues to provide an expendable
cation that facilitates the excreting of titratable acids while
conserving sodium and potassium ions. The increased
Na+/H+ exchanger activity also promotes the
tubular reabsorption of HCO-ketoglutarate generated from glutamine is converted to glucose.
This process generates 2 HCO
-ketoglutarate. The increase in basolateral
Na+-3HCO
Thus the characterization of the molecular mechanisms that regulate expression of the GA and PEPCK genes and the associated signal transduction pathway should provide a paradigm for understanding how the renal proximal convoluted tubule senses changes in acid-base balance and mediates the cell-specific regulation of gene expression.
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LLC-PK1-FBPASE+ CELLS |
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Molecular analyses of the mechanisms that mediate the enhanced
expression of the GA and PEPCK genes during metabolic acidosis required
the identification of a renal cell line that retained the pH-responsive
adaptations. LLC-PK1 cells exhibit a number of properties
that are characteristic of renal proximal tubular cells
(23), including a pH-responsive increase in glutamine metabolism (24, 28). However, LLC-PK1 cells
are unable to synthesize glucose from lactate or pyruvate due to a lack
of FBPase (29). A gluconeogenic subline was isolated by
initially adapting LLC-PK1 cells to low-glucose medium
(<0.5 mM) and then selecting them with essentially glucose-free medium
supplemented with 10 mM sodium pyruvate. The cells that
survived and replicated in the glucose-free selection medium expressed
significant FBPase activity when subsequently grown in either the
absence or presence of 5 mM glucose. Therefore, the isolated
gluconeogenic strain was designated as
LLC-PK1-FBPase+ cells (26). The
selected cells also exhibit a 10-fold higher level of PEPCK activity
than the parental LLC-PK1 cells (36). Net
glucose synthesis from precursor substrates could not be detected. However, treatment of LLC-PK1-FBPase+ cells
growing on pyruvate, oxaloacetate, or -ketoglutarate with 3-mercaptopicolinic acid, a specific inhibitor of PEPCK
(26), caused cell lysis and death. However,
3-mercaptopicolinic acid had no effect on cells grown on
dihydroxyacetone or glycerol, substrates that enter the gluconeogenic
pathway after PEPCK. Thus LLC-PK1-FBPase+ cells
are capable of sufficient gluconeogenesis to grow and replicate in the
absence of sugars, and PEPCK activity is essential when grown on
metabolites that enter the gluconeogenic pathway before the PEPCK reaction.
The parental LLC-PK1 cells exhibit a slow rate of glutamine
catabolism that is only increased slightly when they are grown in
acidic medium (24, 28). This increase occurs without an adaptive increase in mitochondrial GA activity (28). In
contrast, LLC-PK1-FBPase+ cells exhibit an
enhanced oxidative metabolism, an increased mitochondrial density
(25), and a parallel increase in basal GA activity. As a
result, the LLC-PK1-FBPase+ cells exhibit an
enhanced rate of glutamine catabolism (26) and a higher
basal rate of ammonia production (27). Most importantly, when transferred to acidic medium (pH 6.9, 9 mM
HCO
The primary difference between the cell culture system and the
well-characterized pathway of rat renal glutamine catabolism is the
fate of the amine nitrogen. In rat kidney, the glutamate generated by
mitochondrial GA during acidosis is deaminated by GDH, thereby
generating two ammoniums ions per glutamine. In contrast, in both the
LLC-PK1 (28) and
LLC-PK1-FBPase+ (58) cells, the
resulting glutamate is primarily transaminated to pyruvate to form
alanine and -ketoglutarate. This pathway yields only one ammonium
ion per glutamine.
LLC-PK1-FBPase+ cells express both the cytosolic and the mitochondrial forms of PEPCK mRNAs (36). However, when cells were incubated in acidic media for 18 h, the level of the cytosolic PEPCK mRNA was increased threefold whereas the level of the mitochondrial PEPCK mRNA was unchanged. Rat kidney contains two GA mRNAs, a 4.7-kb and a less abundant 3.4-kb mRNA, that are coordinately affected in response to changes in acid-base balance (41, 42). In LLC-PK1-FBPase+ cells, the primary GA mRNAs are 5.0 and 4.5 kb in length (60). However, only the 4.5-kb GA mRNA is increased (3-fold) when LLC-PK1- FBPase+ cultures were incubated with acidic culture media. This increase correlates with the increase in assayable GA activity. The 5.0-kb GA mRNA is constitutively expressed and remains unaltered (27, 60). The two GA mRNAs are produced by alternative splicing of a single transcript (Taylor L and Curthoys NP, unpublished observations). The resulting mRNAs contain distinct 3'-nontranslated regions and encode GA isoforms that have different COOH-terminal sequences. The 3'-nontranslated region of the 5.0-kb porcine GA mRNA lacks the eight-base AU sequence that mediates the pH-responsive stabilization of the rat GA mRNA (60). However, the 4.5-kb GA mRNA, which is the homolog of the human GAc mRNA (14), contains two separate pH-response elements. This finding is consistent with the observation that the apparent half-life of the 4.5-kb GA mRNA was increased 2.3-fold when LLC-PK1-FBPase+ cells were transferred to acidic media (27, 60). Thus the LLC-PK1-FBPase+ cells effectively model the adaptive changes in both PEPCK and GA mRNAs that are observed in rat renal proximal tubular cells. A pH-induced increase in the levels of GDH mRNA also occurs in LLC-PK1-FBPase+ cells (46).
The LLC-PK1-FBPase+ cells exhibit a pleiotropic
phenotype compared with the parental LLC-PK1 cells. In
addition to being gluconeogenic and pH responsive, the
LLC-PK1-FBPase+cells also exhibit a greater
apical proton secretion (27). This correlates with
increased levels of the NHE3 mRNA that encodes the apical
Na+/H+ exchanger (Feifel E and Gstraunthaler G,
unpublished observations). LLC-PK1-FBPase+
cells cultured on permeable supports generate an apical negative transepithelial potential difference of 1.5 mV, whereas
LLC-PK1 epithelia produce an apical positive
transepithelial potential difference. The observed difference results
from different transepithelial ion permeabilities.
LLC-PK1-FBPase+ epithelia are cation selective,
whereas parental LLC-PK1 monolayers are primarily anion
selective (40). More recently, another proximal tubule-specific enzyme, diaminoxidase, and its mRNA were detected uniquely in LLC-PK1-FBPase+ cells
(Wilflingseder D, Gstraunthaler G, and Schwelberger H, unpublished
observations). However, LLC-PK1-FBPase+ cells
do not express alkaline phosphatase activity (25).
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STABILIZATION OF GA MRNA |
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In the rat, the onset of acidosis leads to a gradual and cell-specific increase in the activity of mitochondrial GA. The 7- to 20-fold adaptation within the proximal convoluted tubule requires 5-7 days (11). Previous studies indicate that the increase in GA protein is due to an increase in its relative rate of synthesis (80), which correlates with an increase in the level of translatable (81) and total (41) GA mRNA. The proximal promoter region of the rat renal GA gene lacks an identifiable TATA box (79). Computer analysis of the initial 2.3-kb segment of the promoter identified a number of putative binding sites for known transcription factors that may contribute to basal and activated transcription. However, nuclear run-off assays indicated that the rate of transcription of the renal GA gene is unaffected by alterations in acid-base balance (41, 42). These observations suggest that the increase in GA activity during chronic acidosis may result from an increased stability of the GA mRNA. The 3.4- and 4.7-kb GA mRNAs found in rat kidney are produced by the use of alternative polyadenylation sites. The levels of the two mRNAs are coordinately affected in response to changes in acid-base balance (41, 42).
The selective stabilization of the GA mRNA was initially demonstrated
by stable transfection of various -globin (
G) constructs into
LLC-PK1-FBPase+ cells (31).
Expression of p
G produced a high level of a very stable mRNA
(t1/2 >30 h) that was not affected by
transfer of the cells to acidic medium. In contrast, p
G-GA, which
includes the 956-base segment of 3'-nontranslated sequence that is
contained in both GA mRNAs, was expressed at significantly lower
levels. The decreased expression resulted from the more rapid turnover (t1/2 = 4.6 h) of the
G-GA
mRNA. Transfer of the latter cells to acidic medium resulted in a
pronounced stabilization (6-fold) and a gradual induction of the
G-GA mRNA. These studies indicated that the 3'-nontranslated segment
contains a pH-response element (pH-RE).
Experiments using additional chimeric G constructs indicated that a
340-base segment of the GA mRNA, termed R-2, retained most of the
functional characteristics of the 3'-nontranslated region
(51). Mapping studies, using RNA gel-shift assays,
demonstrated that the specific binding site within the R-2 RNA
consisted of a direct repeat of an eight-base AU sequence.
Site-directed mutation of the direct repeat of the eight-base AU
sequence completely abolished the pH-responsive stabilization of the
G-GA mRNA (49). A
G reporter construct that
contained the 3'-nontranslated region of the PEPCK mRNA, p
G-PCK, was
designed to further test the function of the AU element. When expressed
in LLC-PK1-FBPase+ cells, the half-life of the
G-PCK mRNA was only slightly stabilized (1.3-fold) by growth in
acidic medium. However, insertion of short segments of GA cDNA
containing either the direct repeat or a single eight-base AU-sequence
was sufficient to impart a fivefold pH-responsive stabilization to the
chimeric mRNA. Thus either the direct repeat or a single copy of the
eight-base AU sequence is both necessary and sufficient to function as
a pH-RE.
The apparent binding to the pH-RE is increased threefold in cytosolic extracts prepared from LLC-PK1-FBPase+ cells that were grown in acidic medium (50). Extracts prepared from the renal cortex of rats that were made acutely acidotic also exhibit a similar increase in binding to the direct repeat of the pH-RE. The time course for the increase in binding correlates with the temporal increase in GA mRNA. Scatchard analysis indicated that the increased binding is due to an increase in both the affinity and the maximal binding of the pH-RE binding protein.
A biotinylated oligoribonucleotide containing the pH-RE was used as an
affinity ligand to purify a 36-kDa protein from rat renal cortex
(76). The isolated protein retained the same specific binding properties as observed with crude cytosolic extracts. Microsequencing of the purified protein by mass spectroscopy identified eight peptides that are contained in mouse -crystallin/NADPH:quinone reductase. In addition, three peptides that differed by a single amino
acid from sequences found in mouse
-crystallin/NADPH:quinone reductase were identified. The observed differences may represent substitutions found in the rat homolog. Specific antibodies to
-crystallin/NADPH:quinone reductase blocked formation of the complex
produced by the pH-RE and the purified protein. The 3'-nontranslated region of the GDH mRNA contains four 8-base AU-rich segments in which 7 of the 8-bases are identical to the pH-RE sequence identified in the GA
mRNA. The purified
-crystallin/NADPH:quinone reductase binds to two
of the sequences with high affinity and specificity (Schroeder J, Liu
W, and Curthoys NP, unpublished observations). Furthermore, a chimeric
G-GDH mRNA exhibits a pH-responsive stabilization when stably
expressed in LLC-PK1-FBPase+ cells. Thus
increased binding of
-crystallin/NADPH:quinone reductase to the GA
and GDH mRNAs may initiate their stabilization and increased expression
during acidosis.
-Crystallin/NADPH:quinone reductase constitutes 10% of the total
protein present in the lens of hystricomorph rodents (64) and camelids (18). In these species, the
-crystallin
gene contains an alternative promoter that accounts for its
lens-specific overexpression (21, 52). Similar to other
lens crystallins with a limited phylogenetic distribution,
-crystallin also has a catalytic activity and is expressed at
enzymatic levels in various tissues of different species (20,
65).
-Crystallin possesses a novel NADPH-dependent quinone
oxidoreductase activity that reduces various quinones through the
sequential transfer of single electrons (64).
-Crystallin/ NADPH:quinone reductase was not previously known to
function as an RNA binding protein. However, bovine
-crystallin also
binds with high affinity to Z-DNA and to single-strand DNA (17,
63). This interaction is effectively competed by NADPH, suggesting that the DNA may interact with the dinucleotide binding site. Similar interactions have been reported for other enzymes that
utilize pyridine nucleotides as a substrate, including
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate
dehydrogenase, GDH, thymidylate synthetase, dihydrofolate reductase,
and catalase (35, 63). GAPDH has also been reported to
bind to AUUUA-rich sequences within the 3'-nontranslated regions of
mRNAs. Thus the nucleotide binding site of metabolic enzymes may
function both in catalysis and in gene regulation (35).
The cumulative data are consistent with the following model (Fig.
2). In normal acid-base balance, the
pH-RE present in the 3'-nontranslated region of the GA or GDH mRNA
recruits a site-specific endoribonuclease. The onset of metabolic
acidosis causes an increase in the binding affinity of
-crystallin/NADPH:quinone reductase for the pH-RE. This, in turn,
confers increased protection to the GA mRNA from endonucleolytic
cleavage and results in an increased stabilization of the GA mRNA. This
hypothesis represents the simplest interpretation of the available
data. However, the present data do not rule out more complex
mechanisms. Further characterization of the actual mechanism will be
facilitated through the development of in vitro mRNA decay assays and
the use of recombinant
-crystallin/NADPH:quinone reductase.
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ACTIVATION OF PEPCK TRANSCRIPTION |
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The increase in the level of PEPCK mRNA in rat kidney is initiated within 1 h after onset of acute acidosis and reaches a maximum within 7 h at a level that is sixfold greater than normal (41). The sixfold induced level of PEPCK mRNA is sustained in rats that are made chronically acidotic for periods up to 7 days (42). The observed changes in PEPCK mRNA levels are closely correlated with earlier data that measured changes in the relative rates of PEPCK protein synthesis in normal and acidotic rats (45). Thus regulation of the translation of the PEPCK mRNA is unlikely to contribute to the observed changes in PEPCK gene expression. Furthermore, the apparent t1/2 for the renal PEPCK protein (~6 h) is not altered during acidosis. However, transcription run-off experiments using isolated rat renal nuclei indicated that transcription of the PEPCK gene was rapidly activated after the onset of acute acidosis (41). The initial increase in the relative rate of transcription both preceded and exceeded the initial increase in PEPCK mRNA. Therefore, activated transcription can fully account for the initial induction of PEPCK mRNA.
The cytosolic form of PEPCK is expressed predominantly in liver,
kidney, and adipose tissues (32). Transcription of PEPCK is suppressed during fetal development but is dramatically induced in
liver at birth. Transcription of the PEPCK gene in the postnatal liver
is stimulated by glucagon via cAMP, thyroid hormone, retinoic acid, and
glucocorticoids, whereas insulin and phorbol esters inhibit its
expression. Expression of a chimeric PEPCK-bovine growth hormone gene
in transgenic mice revealed that a relatively small region within the
PEPCK promoter (460 to +73 bp) contains most of the information
required for conferring the appropriate pattern of developmental,
hormonal, and dietary regulation of the PEPCK gene in liver (56,
57). A larger 2-kb segment of the PEPCK promoter was required to
drive expression of the transgene in adipose tissue (57,
71). However, both of these constructs were expressed at
relatively low levels in the kidney.
Confluent and well-differentiated cultures of
LLC-PK1-FBPase+ cells exhibit a threefold
increase in PEPCK mRNA when transferred to acidic medium for
16 h (37). Similar cultures transfected with
PEPCK490- chloramphenicol acetyltransferase (CAT) exhibit a two- to threefold increase in CAT activity when shifted to
acidic medium for 48 h. Mutation of the P2 promoter element that
binds HNF-1 caused an eightfold decrease in basal CAT activity but did
not affect the pH response. In contrast, mutation of the P3(II) element
that contains an AP-1 site or the CRE-1 site had little effect on basal
activity, but each caused a 50% decrease in the pH response. Deletions
or mutations of the other well-characterized elements of the PEPCK
promoter had no effect on either activity. These results suggested that
changes in the activity or levels of proteins in renal proximal tubular
epithelial cells that bind to the P3(II) and CRE-1 elements may mediate
the increased transcription of the PEPCK gene during metabolic
acidosis. Cassuto et al. (5) used a
PCK
597CAT construct along with various deletions and
site-specific mutations to reexamine this question. They again found
that the wild-type construct exhibited a 2.5-fold increase in CAT
activity when the cells were transferred to acidic medium. However,
this response was retained in constructs that lacked the entire P3 and
P4 region or contained a mutation in the P1 element. A mutation in
CRE-1 again caused a partial reduction in the fold-response but this
was due largely to an increase in basal activity. They again observed
that mutation of the P2 element significantly reduced basal activity.
Because they also observed that the P2 mutant had a lower pH response
(1.6-fold), they concluded that the binding of HNF-1 to this element
may contribute to both basal and pH-responsive activation of PEPCK
expression in kidney cells. Finally, Drewnowska et al.
(12) compared the pH responsiveness of
PCK-490CAT and PCK
2300CAT constructs in
subconfluent LLC-PK1- FBPase+ cells. They
reported that a significant pH-response was observed only with the
construct containing the longer promoter element.
Given the divergence of these results and the marginal sensitivity of
the CAT-reporter constructs, the various promoter segments were cloned
into pGL2-Basic (Promega), which encodes the firefly luciferase gene
(Wall Q-T and Curthoys NP, unpublished observations). The activity of
the resulting PCK2300Luc construct in
LLC-PK1-FBPase+ cells is activated 30-fold by
coexpression of the catalytic subunit of PKA (53). This
response maps primarily to the CRE-1 element and is mediated, in part,
by C/EBP
. In contrast, neither PCK
490Luc
nor PCK
2300Luc exhibited a pH-responsive activation.
Stable cell lines that express integrated copies of the PEPCK
promoter/luciferase gene were also isolated and tested for a pH
response. Again, none of these constructs exhibited a pH-responsive
increase in luciferase activity even though the level of the endogenous
PEPCK mRNA was still increased threefold after transfer to acidic
medium. Similar experiments were performed using adenovirus constructs
to infect LLC-PK1-FBPase+ cells with the
PCK
2300Luc gene. Control experiments using a green
fluorescent protein construct indicated that nearly all the
LLC-PK1-FBPase+ cells are rapidly infected with
the adenovirus. This protocol also produced very high luciferase
activity in the LLC-PK1-FBPase+ cells, but
again this activity was not pH responsive. Therefore, elements outside
the proximal promoter may also be involved in this response.
A PEPCK transgene developed by Eisenberger et al. (13)
exhibits a normal developmental profile and appropriate adult levels of
expression in kidney. This construct contains the entire rat PEPCK gene
including 362 bp of the 5'-promoter region and 0.5 kb of the
3'-flanking sequence. It differed from the normal rat gene only by the
removal of a 465-bp EcoRI/SphI fragment from the
exon encoding the 3'-nontranslated region and insertion of the
corresponding segment from the chicken PEPCK gene. In contrast, transgenes that used longer promoter segments to drive expression of a
reporter gene were weakly expressed in kidney (56, 57, 71). Furthermore, the level of the hybrid rat and chicken PEPCK mRNA was increased in the kidneys of the transgenic mice that were made
acidotic (Reshef L, personal communication). These observations strongly suggest that an element downstream of the basal promoter is
essential for normal expression of the PEPCK gene in kidney cortex.
More recently, Cissel and Chalkley (8) mapped the
nuclease-hypersensitive sites within the chromatin structure of the
PEPCK gene in rat kidney and in NRK52E cells, a rat kidney cell line
that does not express the PEPCK gene. The 6,200-,
4,800-,
1,240-,
and +4,650-bp hypersensitive sites previously identified in rat liver
DNA (44) were not present in kidney DNA. In contrast, DNA
from normal and acidotic rats exhibited hypersensitive sites at
3,100,
400/+1, +1,900, and +6,200 bp. However, the
3,100- and
+6,200-bp sites were also present in DNA isolated from the NRK52E
cells. Thus only the proximal promoter (
400/+1) region and the unique
site at +1,900 bp correlated with kidney-specific expression of the PEPCK gene. The latter site is contained within intron 4 of the rat
PEPCK gene. This site may contain the additional element that is needed
for enhanced expression and pH-responsive induction in kidney cells.
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SIGNAL TRANSDUCTION |
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A decrease in pHi must initiate a signal that mediates
the increase in transcription of the PEPCK mRNA. The potential
involvement of known mitogen-activated protein kinase (MAPK) activities
[extracellular signal-regulated kinse (ERK)1/2, c-Jun
NH2-terminal kinase (JNK), p38 stress-activated protein
kinase (SAPK)] was examined by determining the effects of specific
MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA
levels (Feifel E, Euler S, Obexer P, Tang A, Wei Y, Schramek H,
Curthoys NP, and Gstraunthaler G, unpublished observations). The
protein synthesis inhibitor, anisomycin, is a potent activator of the
p38 SAPK. Anisomycin increased PEPCK mRNA to levels comparable to those
observed with acid stimulation. Transfer of
LLC-PK1-FBPase+ cultures to acidic medium
resulted in phosphorylation, and thus activation, of p38 SAPK. ATF-2 is
a basic leucine zipper transcription factor that activates
transcription after dual phosphorylation by p38 SAPK. Phosphorylation
of ATF-2 was also observed in LLC-PK1-FBPase+
cells after treatment with acidic medium. SB-203580, a specific p38
SAPK inhibitor, produced a dose-dependent inhibition of both the acid-
and the anisomycin-mediated induction of PEPCK mRNA and blocked
phosphorylation of ATF-2. In contrast, the ERK1/2 inhibitors PD-098059
and U-0126 did not affect basal or acid-induced PEPCK mRNA levels.
Similarly, the JNK-specific inhibitor curcumin had no effect. In
addition, JNK phosphorylation and JNK activity were decreased in
acid-adapted cells, indicating that neither ERK1/2 nor JNK plays a
significant role. Of the four known p38 SAPKs, only the SB-sensitive
-isoform is strongly expressed in LLC-PK1-FBPase+ cells. The octanucleotide
sequence of the CRE-1 site of the PEPCK promoter (TTACGTCA) is a
perfect match for the consensus element for binding ATF-2
(6). Gel-shift analysis using a labeled oligonucleotide containing the CRE-1 element produced a band that was partially supershifted with antibodies specific for ATF-2. Thus the SB-sensitive p38
SAPK/ATF-2 signaling pathway may contribute to the pH-responsive induction of PEPCK mRNA transcription in renal
LLC-PK1-FBPase+ cells.
On the basis of existing data, the following model (Fig.
3) was developed as a hypothesis
for the mechanism by which PEPCK mRNA transcription is induced in the
renal proximal convoluted tubule during metabolic acidosis. During
normal acid-base balance, only the P2 and possibly the P3(II) regions
of the PEPCK promoter are occupied with bound transcription factors
(5). A decrease in pHi leads to activation of
the -isoform of p38 SAPK that, in turn, phosphorylates and activates
ATF-2. The activated ATF-2 binds to the CRE-1 element and recruits or
interacts with an additional transcription factor that binds downstream
of the promoter. The resulting complex recruits the appropriate
coactivator and polymerase that are necessary for transcriptional
activation of the PEPCK gene.
|
The pH-responsive stabilization of the GA mRNA and the activated
transcription of the PEPCK gene are coordinately regulated within the
renal proximal tubule. Therefore, the same signal transduction mechanism is likely to initiate both adaptations. However, the pH-responsive increase in GA mRNA levels in
LLC-PK1-FBPase+ cells is not blocked by
SB-203580 (16). Thus the enhanced binding of
-crystallin/NADPH:quinone reductase to the GA mRNA may be mediated
by a kinase that is upstream of p38 SAPK (Fig. 3A). This model provides a hypothesis to further characterize the molecular events that allow the renal proximal convoluted tubule cells to sense
changes in pH and mediate a response that leads to increased transcription of specific genes and the selective stabilization of
certain mRNAs.
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ACKNOWLEDGEMENTS |
---|
This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37124 and DK-43704 (to N. P. Curthoys) and Austrian Science Fund Grants P11126 and P12705 (to G. Gstraunthaler).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: N. P. Curthoys, Dept. of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870 (E-mail: ncurth{at}lamar.colostate.edu).
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