Differential expression and acid-base regulation of
glutaminase mRNAs in gluconeogenic
LLC-PK1-FBPase+ cells
Gerhard
Gstraunthaler1,
Thomas
Holcomb2,
Elisabeth
Feifel1,
Wenlin
Liu2,
Nikolaus
Spitaler1, and
Norman P.
Curthoys2
1 Institute of Physiology, University of
Innsbruck, A-6010 Innsbruck, Austria; and
2 Department of Biochemistry and Molecular
Biology, Colorado State University, Fort Collins, Colorado
80523-1870
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ABSTRACT |
LLC-PK1-FBPase+ cells, which are
a gluconeogenic substrain of porcine renal LLC-PK1
cells, exhibit enhanced oxidative metabolism and increased levels of
phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic
medium (pH 6.9, 9 mM HCO
3), LLC-PK1-FBPase+ cells also exhibit a greater
increase in ammonia production and respond with an increase in
assayable PDG activity. The changes in PDG mRNA levels were examined by
using confluent cells grown on plastic dishes or on permeable membrane
inserts. The latter condition increased the state of differentiation of
the LLC-PK1-FBPase+ cells. The levels of the
primary porcine PDG mRNAs were analyzed by using probes that are
specific for the 5.0-kb PDG mRNA (p2400) or that react equally with
both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish-
and filter-grown LLC-PK1-FBPase+ cells, the
predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic
media. However, in filter-grown epithelia, which sustain an imposed pH
and HCO
3 gradient, this adaptive
increase is observed only when acidic medium is applied to both the
apical and the basolateral sides of the epithelia. Half-life
experiments established that induction of the 4.5-kb PDG mRNA was due
to its stabilization. An identical pattern of adaptive increases was
observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes
were observed in the levels of the 5.0-kb PDG mRNA in either cell
culture system. Furthermore, cultures were incubated in low-potassium
(0.7 mM) media for 24-72 h to decrease intracellular pH while
maintaining normal extracellular pH.
LLC-PK1-FBPase+ cells again responded with
increased rates of ammonia production and increased levels of the
4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis
is the initiator of this adaptive response. Because all of the observed
responses closely mimic those characterized in vivo, the
LLC-PK1-FBPase+ cells represent a valuable
tissue culture model to study the molecular mechanisms that regulate
renal gene expression in response to changes in acid-base balance.
metabolic acidosis; ammoniagenesis; gluconeogenesis; phosphoenolpyruvate carboxykinase; renal cell culture
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INTRODUCTION |
A MAJOR REGULATORY FUNCTION of the kidney is to
maintain acid-base homeostasis by excreting excess acids or anions
primarily in the form of ammonium salts. It is well established that
during metabolic acidosis, the rates of renal ammonia production and excretion are increased significantly (45, 46). The proximal convoluted
tubule is the major site of both the basal formation of urinary ammonia
and the adaptive increase in glutamine catabolism and ammoniagenesis
(9, 11, 13, 49).
The initial reactions in the primary pathway of rat renal glutamine
catabolism and ammoniagenesis are catalyzed by two mitochondrial enzymes, phosphate-dependent glutaminase (PDG) and glutamate
dehydrogenase (GDH) (Fig. 1).
Renal extraction of plasma glutamine occurs through peritubular uptake
into proximal tubular cells and by apical reabsorption via a
Na+-dependent transporter. The glutamine is then
transported into the mitochondria and deamidated by PDG. The resulting
glutamate is deaminated by GDH, thereby generating two ammonium ions
and
-ketoglutarate (Fig. 1). However, in various renal cell culture systems, glutamine is first deamidated by PDG and then the amine nitrogen of glutamate is primarily transaminated to pyruvate to form
alanine and
-ketoglutarate (19, 32, 35). This pathway yields only
one ammonium ion per glutamine. In chronic metabolic acidosis, the
elevated rat renal extraction and catabolism of glutamine is
accomplished, in part, by increasing the levels of the PDG and GDH
enzymes in cells of the proximal convoluted tubule (9, 11, 24, 49).
Expression of the cytosolic phosphoenolpyruvate carboxykinase
(PEPCK), a key regulatory enzyme of gluconeogenesis, is also increased
in this nephron segment (7, 24), suggesting a direct link between the
ammoniagenic and gluconeogenic pathways in proximal epithelial cells
(41, 44, 46).

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Fig. 1.
Primary pathways of proximal tubular glutamine metabolism and renal
ammoniagenesis. Deamidation and deamination of glutamine is catalyzed
by phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase
(GDH), respectively. Alternatively, glutamate can be transaminated by
alanine aminotransferase (ALT) (19). -KG-DH, -ketoglutarate
dehydrogenase.
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Over the past decade, significant progress in understanding the
regulation of renal glutamine and ammonia metabolism has been achieved
through the use of cell and tissue culture techniques (14, 19, 29, 35,
40) and by molecular biological approaches, including the isolation of
the genes for the key regulatory enzymes of glutamine metabolism and
the establishment of specific molecular probes. In this context, a
full-length PDG cDNA was isolated, sequenced, and expressed (43). By
utilizing this molecular probe, the kinetics of adaptation of rat renal
PDG mRNA that occur in response to alterations in acid-base balance
were defined (24, 25, 47). It was shown that the increases in both PDG
and PEPCK mRNAs (51) precede the increase in assayable enzyme
activities in rat renal cortex. Thus the increases in renal PDG and
PEPCK activities (7, 9, 11, 27, 49) are due to an increase in their
relative rates of synthesis, which correlate with an increase in the
levels of their respective mRNAs (24, 25, 42, 47). However, the
mechanisms by which acidosis causes increased expression of the two
genes differ significantly (24, 25). The onset of acidosis causes a
rapid increase in transcription of the PEPCK gene, which in turn
accounts for the increased PEPCK mRNA levels. In contrast, PDG mRNA
increases without altering its rate of transcription, suggesting that
the increase in PDG mRNA results from increased stability of the mRNA
(25). Indeed, a pH-responsive instability element in the
3'-untranslated region of rat PDG mRNA and a specific
mRNA-binding protein were recently identified (21, 30). Thus
transduction of the initial stimuli produced by acidosis may activate
divergent, but temporally coordinated, mechanisms to cause the observed
increase in specific mRNAs. However, the signals triggering these
adaptive changes, as well as the specific molecular downstream events,
are unknown. The characterization of this pathway will require a
gluconeogenic and pH-responsive renal cell culture system that can
serve as an effective model.
We previously isolated LLC-PK1-FBPase+ cells, a
gluconeogenic substrain of porcine LLC-PK1 renal epithelial
cells (16). LLC-PK1-FBPase+ cells exhibit
enhanced oxidative metabolism and decreased glycolytic activity (18).
Furthermore, LLC-PK1-FBPase+ cells express
significant levels of the key gluconeogenic enzymes, fructose-1,6-bisphosphatase (16) and the cytosolic and mitochondrial isoforms of PEPCK (22). It was shown that
LLC-PK1-FBPase+ cells express high levels of
PDG and PEPCK mRNAs (29), which are regulated by culture medium pH with
kinetics similar to those observed in vivo in the rat kidney (24). In
addition, we recently found that the activity and mRNA levels of only
the cytosolic isoform of PEPCK are increased in response to acidosis in
these cells (22).
Previous studies on the regulation of PDG mRNA in
LLC-PK1-FBPase+ cells (29) used slot-blot
assays and a rat cDNA probe, pGA13, for which the sequence similarity
to the porcine PDG mRNA was unknown. Therefore, to explore in more
detail the expression of PDG mRNA in
LLC-PK1-FBPase+ cells, a homologous porcine PDG
cDNA was cloned (termed pGA201) (38) by screening a
LLC-PK1
-gt11 cDNA library with the rat PDG cDNA, pGA104
(43). Specific probes derived from the porcine PDG cDNA were then used
to characterize the expression of multiple PDG mRNAs in
LLC-PK1-FBPase+ cells. Two distinct PDG mRNAs,
which are 5.0 and 4.5 kb in length, were clearly expressed in
subconfluent cells (38).
In the present study, the expression and acid-base regulation of the
two primary glutaminase transcripts were characterized in confluent
LLC-PK1-FBPase+ monolayer cultures and in
epithelia grown on permeable tissue culture inserts to test the
potential effect of increasing the state of differentiation of the
cells (17, 20, 32, 48). We observed that only the 4.5-kb PDG mRNA is
increased (3-fold) after adaptation to acidic media for 18 h. However,
in filter-grown epithelia, this adaptive response occurs only when
acidic medium is applied to both the apical and the basolateral sides.
Furthermore, experiments using low-potassium medium indicate that a
decrease in intracellular pH is the apparent initiator of this adaptive response.
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MATERIALS AND METHODS |
Cell culture.
Gluconeogenic LLC-PK1-FBPase+ cells (16, 18,
22) were cultured in DMEM with 5.5 mM D-glucose, 2 mM
L-glutamine, and 26 mM NaHCO3 (pH 7.5),
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Culture media were prepared from DMEM-base
(D-5030, Sigma Chemical, St. Louis, MO) as described previously (19,
22). Acidic medium (pH 6.9) contained 9 mM NaHCO3 and was
supplemented with the appropriate amount of NaCl to maintain equivalent
osmolarity (19). Low-potassium DMEM was prepared from MEM amino acid
solution (M-7020, Sigma Chemical) and MEM vitamin solution (M-6895,
Sigma Chemical) as basal components, supplemented with glucose, sodium
pyruvate, and inorganic salts without KCl. The potassium concentration
of medium after addition of 10% fetal bovine serum was determined by
flame photometry. Cultures were incubated at 37°C in a 5%
CO2-95% air atmosphere. Routinely, cultures were fed three
times a week. Experimental cultures were always fed 24 h before
adaptation. Confluent monolayers were subcultured (split ratio 1:3) by
using 0.25% trypsin and 0.02% EDTA in Ca2+- and
Mg2+-free buffered saline.
Cultures were grown for 10-12 days to produce confluent monolayers
in 10-cm plastic tissue culture dishes (Falcon Optilux, no. 3003) by
using 10 ml of culture medium or as epithelial cultures on microporous
tissue culture inserts (Nunc TC Inserts, A/S Nunc). Filter inserts (3 inserts/experiment) were placed in 10-cm dishes with an excess of
basolateral culture media (15 ml) and an apical medium volume of 2.5 ml.
Adaptation protocols and biochemical assays.
Cultures were adapted to metabolic acidosis by switching to acidic
media (pH 6.9) for the indicated times. Identical protocols were used
for low-potassium adaptation. In previous studies it was shown that
gradual decreases of culture medium pH from 7.6 to 6.8 resulted in a
gradual increase in the ammoniagenic response in LLC-PK1
cells (19) and in intermediate changes in PDG and PEPCK mRNA levels in
LLC-PK1-FBPase+ cells (29). Therefore, in the
present study, culture conditions (pH 6.9, 9 mM
HCO
3) that slightly exceed a
physiological metabolic acidosis were used to maximize the response for
Northern analysis (see below). PDG enzyme activity was determined in
cell homogenates as described elsewhere (18). Ammonia was analyzed enzymatically in culture media samples by established methods (19).
Northern analysis.
For each experiment cells were harvested from either a 10-cm dish or
three filter inserts. Total RNA was isolated by using the acid
guanidium thiocyanate method as described in detail (22, 31, 38).
Formaldehyde-agarose gel electrophoresis, transfer to GeneScreen Plus
membranes (NEN; New England Nuclear), hybridization and
posthybridization washings of blots were carried out as described previously (22, 23, 31, 38). Blots were exposed by using either a
PhosphorImager Screen (Molecular Dynamics) or autoradiographic film
(Kodak BioMax MS). Quantitation of mRNA levels was accomplished using a
by PhosphorImage Analyzer or a Personal Densitometer SI-Scanner (Molecular Dynamics). Exposure times varied from 48 h for
PhosphorImager Screens to 5-7 days for films of PEPCK blots and
10-12 days for films of PDG blots. Sample integrity and equal
loading of 20 µg RNA/lane were monitored by staining with ethidium
bromide after electrophoresis. In addition, some blots were also
stripped of the initial probe and rehybridized with a human
-actin
cDNA (Clontech Laboratories) (31). For the half-life determinations,
RNA polymerase II-dependent transcription was blocked by adding 65 µM
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB) as
described in detail (21, 23).
cDNA probes.
The glutaminase cDNA probes used for hybridization include sequences
that correspond to the coding region of rat PDG mRNA (r1500), the
coding region of both porcine PDG mRNAs (p930), and the
3'-untranslated region of the 5.0-kb porcine PDG mRNA (p2400) (Fig. 2). Probe r1500 is an Acc I
restriction fragment of pGA104 (43), and p2400 is an Nhe
I/Not I restriction fragment of pGA201 (38). The 4.5- and the
5.0-kb PDG mRNAs were separated and purified from total
LLC-PK1-FBPase+ RNA by selective cleavage with
RNase H and chromatography on oligo(dT)-cellulose (37). A
1.1-kb segment of coding sequence was PCR amplified and sequenced from
the two purified mRNAs by using the same set of primers. The two
sequences were identical, and they have a 92% identity with a segment
(574-1671 bp) of the rat PDG cDNA (43). The PCR product was cloned
into pBluescript SK(-) (Stratagene) and the p930 probe was isolated by
restricting with Kpn I and EcoR I (C. Curtis and N. P. Curthoys, unpublished data). For probing PEPCK mRNA, a 1.6-kb
Bgl II fragment of pPCK-10 (51, 52), which encodes the rat
cytosolic PEPCK, was used. The pPCK-10 plasmid was kindly provided by
Dr. R. Hanson (Case Western Reserve Univ.).

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Fig. 2.
PDG mRNAs in rat kidney and in LLC-PK1-FBPase+
cells and corresponding cDNA probes used in the present study. Regions
of the PDG mRNAs that have been cloned and sequenced from rat kidney
and from porcine LLC-PK1-FBPase+ cells are
drawn to scale as solid lines. Dashed lines represent approximate
lengths of regions that have not been cloned. Sections of cDNAs used as
probes are shown below their corresponding mRNAs. Probes are designated
by species (r, rat; p, porcine) and by length (in bp). Thus p2400 is a
cDNA containing 3'-untranslated sequence that hybridizes
specifically to the porcine 5.0-kb PDG mRNA, whereas r1500 and p930 are
probes from the coding region of porcine and rat PDG mRNAs,
respectively, that hybridize to both the 4.5- and the 5.0-kb PDG mRNAs
from LLC-PK1-FBPase+ cells. AUG, start codon;
UAA, stop codon; pA, putative polyadenylation site.
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Statistical analysis of results was performed using the unpaired
Student's t-test.
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RESULTS |
Enhanced oxidative metabolism and ammoniagenesis of
LLC-PK1-FBPase+
cells and their biochemical response to metabolic acidosis.
LLC-PK1-FBPase+ cells were selected by growing
the parental LLC-PK1 cells in the absence of added glucose
(16). The selected cells express the key gluconeogenic enzymes
fructose-1,6-bisphosphatase and cytosolic PEPCK (18, 22, 23, 31) and
thus exhibit gluconeogenic competence. In addition,
LLC-PK1-FBPase+ cells exhibit an increased
oxidative metabolism of glutamine (16), another key feature of renal
proximal convoluted tubular cells. As shown in Fig.
3, the increased basal catabolism of
glutamine is manifested by an increased mitochondrial volume density
(18) that is paralleled by increased basal activity of the
mitochondrial phosphate-dependent glutaminase (PDG) and higher baseline
rates of ammonia production.

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Fig. 3.
Absolute mitochondrial volumes (A), PDG enzyme activities
(B), and baseline ammonia production rates (C) of
wild-type LLC-PK1 (solid bars) and gluconeogenic
LLC-PK1-FBPase+ cells (hatched bars).
Values are means ± SD of 3-6 experiments. Mitochondrial
volume data are taken from Ref. 18. Enzyme activities were determined
in cell homogenates and are expressed as mU/mg protein
(nmol · min 1 · mg
cell protein 1). Ammonia
production rates are expressed as µmol · mg
protein · 24 h 1 at pH
7.5.
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Furthermore, the LLC-PK1-FBPase+ cells exhibit
a pronounced increase in ammonia generation in response to a decrease
in pH and HCO
3 concentration of the
culture medium (Fig. 4). This adaptive
response was also significantly greater than that observed with
LLC-PK1 cells (19). In addition,
LLC-PK1-FBPase+ cells respond with an increase
in assayable PDG enzyme activity (Fig. 5),
which is not observed in LLC-PK1 wild-type cells (19). Thus
the LLC-PK1-FBPase+ cells produce an adaptation
to treatment with acidic medium that more closely mimics the overall
response to metabolic acidosis that is observed in rat kidney.

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Fig. 4.
Rates of ammonia production by wild-type LLC-PK1 (solid
bars) and gluconeogenic LLC-PK1-FBPase+ cells
(hatched bars) in response to treatment with acidic medium (24-72
h at pH 6.9). Values are means ± SD of 3-6 series of
experiments. Confluent cultures were adapted for 3 consecutive 24 h-intervals in acidic media. After each incubation interval, culture
media were removed and analyzed, and cultures were refed for further
incubation. Metabolic rates are expressed as
µmol · mg protein · 24 h 1. * P < 0.05 and
** P < 0.01 compared with control (pH 7.5).
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Fig. 5.
Time course of induction of assayable PDG enzyme activity in
LLC-PK1-FBPase+ cells during adaptation to
metabolic acidosis. Inset: lack of PDG induction in
LLC-PK1 cells (data taken from Ref. 19). Values are means ± SE with numbers of determinations in parentheses. PDG activity is
expressed as mU/mg protein
(nmol · min 1 · mg
cell protein 1). * P < 0.05, statistically significant increases in enzyme activity compared with
control values.
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Northern analysis of acid-mediated induction of PDG mRNA in dish-
and filter-grown
LLC-PK1-FBPase+
cells.
The levels of the two primary PDG mRNAs, which are 4.5 and 5.0 kb in
length (38), were analyzed by using the porcine and rat cDNA probes,
shown schematically in Fig. 2. The 2.4-kb porcine probe (p2400) encodes
the unique 3'-untranslated region of the 5.0-kb mRNA, whereas the
930-bp porcine cDNA (p930) contains a segment of coding sequence that
is identical in the 5.0- and the 4.5-kb porcine PDG mRNAs. Thus the
former probe is specific for the 5.0-kb PDG mRNA, whereas the latter
reacts equally with both PDG mRNAs. A 1.5-kb rat cDNA probe (r1500),
which corresponds to the coding region of rat renal PDG mRNA, was also
used (Fig. 2, top). The mRNA levels of cytosolic PEPCK were
probed with a specific rat cDNA probe from pPCK-10 (22, 51, 52).
Basal and acid-induced expression of PDG and PEPCK mRNAs were examined
in LLC-PK1-FBPase+ cells grown for 10-12
days to achieve full differentiation on plastic tissue culture dishes
or in epithelial cultures on permeable supports. The latter culture
condition was developed to further increase the state of
differentiation of the polarized epithelial cells (17, 20), which might
augment the adaptive response to metabolic acidosis. Phase-contrast
microscopic examination revealed remarkable differences in cell shape
and cell density of filter-grown
LLC-PK1-FBPase+ epithelia compared with
dish-grown monolayers (Fig. 6). In
addition, acidic media can be applied to both sides of the filter
inserts carrying the epithelial layer or to either the apical or the
basolateral side. In the latter cases, a pH and
HCO
3 gradient is imposed across the
epithelia so that the sidedness of an extracellular signal or receptor
that potentially mediates the pH-response could be determined.

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Fig. 6.
Light-microscopic comparison of LLC-PK1-FBPase+
cultures grown in plastic dishes and on microporous filter inserts.
Native phase-contrast micrographs of dish (A)- and filter-grown
LLC-PK1-FBPase+ cells (B) 12 days after
seeding. Magnification, ×75.
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Filter-grown cultures were incubated in control (pH 7.5) or acidic (pH
6.9) media on both sides, or under conditions with acidic media on
either epithelial side (apical pH 6.9 or basolateral pH 6.9). After 18 h of incubation, the pH of the media was determined (Fig.
7). The pH of media samples incubated
without cells remained constant. Maturation of the cultured epithelium
and functional integrity during the adaptation period was monitored by
measuring the transepithelial potential difference (17, 20). Under
control and acidic conditions (pH 7.5 or 6.9 on both sides),
LLC-PK1-FBPase+ epithelia acidify the apical
compartment, thereby generating a transepithelial pH gradient of
~0.15-0.20 pH units (Fig. 7). This effect is slightly more
pronounced under acidic conditions (Fig. 7, right bars). When
incubated with acidic media on either side,
LLC-PK1-FBPase+ epithelia are able to sustain
the imposed pH gradient for at least 18-24 h (Fig. 7).

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Fig. 7.
pH of culture media in apical (solid bars) and basolateral fluid
compartments (hatched bars) measured 18 h after superimposing pH
gradients over filter-grown LLC-PK1-FBPase+
epithelia. Values are means ± SD of 6 independent series of
experiments using 3 filters/experiment. Control medium contained 26 mM
NaHCO3 (pH 7.5) whereas acidic medium contained 9 mM
NaHCO3 (pH 6.9). Steady-state pH was measured with a
temperature-compensated mini-glass electrode immediately after removal
of cultures from the incubator. basolat., Basolateral; a+b, apical plus
basolateral.
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Representative Northern blots of RNA isolated from acid-adapted dish-
and filter-grown LLC-PK1-FBPase+ epithelial
cells are displayed in Fig. 8. The results
of four to six independent experiments are summarized in Fig.
9. Data from acidic cultures were
normalized to the respective controls. The 4.5-kb PDG mRNA (probed with
r1500 or p930) and the 2.7-kb cytosolic PEPCK mRNA are increased
approximately threefold on adaptation to acidic medium for 18 h in
LLC-PK1-FBPase+ monolayer cells in tissue
culture dishes (Fig. 8, 2nd lane from the left, and Fig. 9, 2nd
bar from the left, respectively). In filter-grown
LLC-PK1-FBPase+ epithelia, however, increases
in PDG and PEPCK mRNAs were only observed when acidic medium was
applied to both the apical and the basolateral sides (Fig. 8,
right lanes; Fig. 9, right bars; apical + basolateral,
pH 6.9). Application of acidic media to either side of the cultured
epithelium (apical pH 6.9 or basolateral pH 6.9) was not sufficient to
induce a significant response. The levels of cytosolic PEPCK mRNA are
somewhat lower in filter-grown LLC-PK1-FBPase+
cells compared with dish-grown cells (Fig. 8, pPCK). However, the
acid-mediated increase, normalized to control filter epithelia, was of
the same magnitude as found in dishes (Fig. 9C). The 5.0-kb PDG
mRNA, which hybridized specifically to the p2400 probe (see Fig. 2),
was unchanged and remained at a constant level under all experimental
conditions (Fig. 8, top).

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Fig. 8.
Representative Northern blots of acid-adapted
LLC-PK1-FBPase+ cells grown in plastic dishes
and on microporous filter inserts. Cells were grown for 10-12 days
to confluence. Effects of decreased extracellular pH were determined by
replacing growth medium with medium containing 9 mM NaHCO3
(pH 6.9) for 18 h. Filter-grown epithelia were adapted by adding acidic
media to either apical, basolateral, or both sides (see Fig. 7). Total
RNA samples (20 µg) were electrophoresed and blotted onto GeneScreen
Plus membranes. Blots were hybridized with the PDG cDNA probes
p2400, r1500, and p930 (Fig. 2) and with the cytosolic
phosphoenolpyruvate carboxykinase (PEPCK) probe, pPCK-10 (22,
51, 52). Equal loading of samples is shown by ethidium bromide-stained
28s RNA bands.
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Fig. 9.
Summary of results of acid adaptation displayed in Fig. 8. A:
PDG probe r1500. B: PDG probe p930. C: PEPCK probe
pPCK-10. Values are means ± SE of 4-6 independent series of
experiments. Data of dish- and filter-grown cells were normalized to
their respective controls.
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Determination of the half-lives of the 4.5-kb PDG mRNA in control
and acidotic
LLC-PK1-FBPase+
cells.
In rat kidney, the in vivo induction of PDG mRNA during metabolic
acidosis is due to an increased stability of the mRNA (21, 25, 47). To
test whether the same mechanism occurs in acid-adapted LLC-PK1-FBPase+ cells, the apparent half-lives
of basal and acid-induced 4.5-kb PDG mRNAs were determined by
quantitating the rates of decrease that occur after inhibition of RNA
polymerase II-dependent transcription with DRB (Fig.
10). Both the control and acid-induced
4.5-kb mRNA bands exhibit first-order decay profiles. However, the
apparent half-lives calculated from the rates of decrease were 3.4 h
for control and 7.8 h for the acid-induced 4.5-kb PDG mRNA. Thus the apparent half-life of the 4.5-kb mRNA is increased 2.3-fold.

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Fig. 10.
Apparent half-lives of 4.5-kb PDG mRNA in control and acidotic
LLC-PK1-FBPase+ cells. Data are means of 4 sets
of experiments. Cultures were grown for 12 days and then maintained for
18 h in either fresh normal (pH 7.5, ) or acidic (pH 6.9, )
media. Total RNA was isolated either immediately before (0 h) or at
various times (3-9 h) after 65 µM
5,6-dichloro-1- -D-ribofuranosylbenzimidazole (DRB) was
added. Levels of 4.5-kb PDG mRNA were detected by using the p930 probe
and quantitated by PhosphorImager analysis. Normalized levels of 4.5-kb
PDG mRNA were plotted as log percentage remaining vs. time after
inhibition of mRNA synthesis by DRB.
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Effects of low-potassium-containing media on ammonia production and
on PDG and PEPCK mRNA levels.
Potassium deficiency causes an extracellular alkalosis and an
intracellular acidosis (1, 5). It also elicits proximal tubular
adaptations similar to those seen in metabolic acidosis (36, 50). On
the basis of these observations, it was hypothesized that the proximal
tubular adaptations are initiated by changes in intracellular pH (45,
46). Confluent LLC-PK1-FBPase+ monolayers were
adapted to low-potassium media for 24-72 h to induce an
intracellular acidosis at normal extracellular pH. The potassium
concentration of the medium was 0.7 mM as determined by flame
photometry, compared with 5.4 mM in control DMEM. However, the pH of
both media was 7.5. As presented in Fig.
11,
LLC-PK1-FBPase+ cells grown in low-potassium
media exhibit an adaptive increase in the rate of ammonia production
that is slightly greater after 48-72 h than that observed with
acidic medium (for comparison, see Fig. 4). Representative Northern
blots are depicted in Fig. 12 and are
summarized in Fig. 13.
LLC-PK1-FBPase+ cells also responded to
incubation in low-potassium media with a threefold increase in the
levels of the 4.5-kb PDG and cytosolic PEPCK mRNAs as occurs on
incubation of cells in acidic media. In contrast to using acidic media,
where maximal adaptation occurs after 18-24 h (Figs. 8 and 9)
(38), the maximal response to low-potassium medium is achieved after 48 h of incubation (Figs. 12 and 13), which is consistent with the
sustained increase in ammonia generation (Fig. 11).

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Fig. 11.
Rates of ammonia production by LLC-PK1-FBPase+
cells in response to treatment with acidic medium (pH 6.9; solid bars)
and low-potassium-containing medium (hatched bars). Values are
means ± SD of 3 independent series of experiments.
Confluent cultures were adapted for 3 consecutive 24 h-intervals in
experimental media. After each incubation interval, culture media were
removed and analyzed, and cultures were refed for further incubation.
Open bar is basal ammonia production rate in control medium (for
comparison, see Fig. 4). Metabolic rates are expressed as µmol/mg
cell protein for each 24-h period. * P < 0.05 and
** P < 0.01 compared with control.
|
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Fig. 12.
Northern analysis of glutaminase and PEPCK mRNA levles in
potassium-depleted LLC-PK1-FBPase+ cultures.
Cells were grown in plastic dishes for 10-12 days and then
incubated for 24-72 h in low-potassium (0.7 mM)-containing culture
media. Acidic cultures in plastic dishes served as positive control.
Total RNA was isolated from each plate, and 20 µg of each sample were
electrophoresed per lane. Porcine PDG mRNA was probed with cDNA p930,
and cytosolic PEPCK was probed with pPCK-10. Equal loading of samples
is shown by the ethidium bromide-stained 28s RNA bands (for further
details see Fig. 8).
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Fig. 13.
Summary of results of potassium-depletion experiments shown in Fig. 12.
Data of acid-adapted and potassium-depleted cells were normalized to
their respective control. Values are means ± SE of 3 series of
experiments.
|
|
 |
DISCUSSION |
In the present study, confluent cultures of the gluconeogenic
LLC-PK1-FBPase+ cells were tested as an in
vitro model system for investigating the molecular mechanisms by which
alterations in acid-base balance affect the levels of mRNAs that encode
key regulatory enzymes of renal proximal convoluted tubular
gluconeogenesis and ammoniagenesis.
In vivo, the adaptive increases in gluconeogenesis and ammoniagenesis
in the renal proximal convoluted tubule of carnivorous and omnivorous
mammals during metabolic acidosis are coordinated to produce an
increased extraction and catabolism of plasma glutamine and an enhanced
excretion of titratable acids. Renal catabolism of glutamine leads to
the generation of ammonium ions and
-ketoglutarate (Fig. 1) (10).
For net acid secretion to occur, the
-ketoglutarate, which is a
divalent anion, must be neutralized by either complete oxidation or
conversion to glucose (44). The flux of
-ketoglutarate through
either pathway also results in the net production and the basolateral
release of 2 mol HCO
3, which partially
compensates for the systemic acidosis (44-46).
Compared with the parental LLC-PK1 cells, the gluconeogenic
LLC-PK1-FBPase+ cells exhibit an increased
oxidative metabolism (18) and an increased rate of glutamine
consumption (16). As depicted in Fig. 3, the
LLC-PK1-FBPase+ cells also exhibit a greater
rate of ammonium ion production that correlates with an elevated basal
PDG activity. Furthermore, the LLC-PK1-FBPase+
cells respond to acidification of the culture medium with a pronounced increase in ammonium ion production (Fig. 4) that again correlates with
a similar increase in assayable glutaminase enzyme activity (Fig. 5). Although other proximal tubule-like renal cell lines, such as the parental LLC-PK1 cells and opossum
kidney cells, also exhibit slight increases in glutamine metabolism
after exposure to acidic medium, such cells are primarily glycolytic
and catabolize glutamine at significantly lower rates (15, 19).
Furthermore, LLC-PK1 wild-type cells lack any adaptive
increase in glutaminase activity (Fig. 5, inset; Ref. 19).
During the past decade, renal epithelial cell and tissue cultures have
emerged as a powerful tool to study in vitro several aspects of renal
metabolism, epithelial transport, and renal cell growth and
differentiation (14, 17). Modern cell and tissue culture techniques
enable renal epithelial cells to grow and be maintained at a state of
differentiation, comparable with the in vivo tissue. The use of cell
biological, immunological, and molecular biological methods has opened
new avenues in physiological and pharmacological research. Thus the
advantages of using cultured cells in in vitro studies of renal
metabolism are obvious and manifold. Easy manipulation of the cells and
the ability to change tissue culture parameters individually, in
combination, or sequentially in short- or long-term applications have
established cultured renal epithelia as valuable tools for
investigating renal cell metabolism and function at the cellular and
subcellular level. Such studies can also be accomplished without
inducing higher ordered regulatory mechanisms as in complex organisms.
Continuous renal epithelial cell lines as well as renal proximal
tubular primary cultures from a variety of mammalian species including
human have been used to study renal gluconeogenesis and ammoniagenesis
in vitro (14, 15, 19, 22, 29, 35). Most of the proximal tubular primary
cultures were established to study renal proximal tubular transport
functions and hormone responsiveness. Some of the primary cultures were
also initiated to study in vitro proximal tubular metabolic
features, such as gluconeogenesis, pH-mediated ammoniagenesis, and the
expression and regulation of PEPCK and PDG. However, these efforts met
with limited success.
In the present study, experiments were performed on 10-12 day
confluent LLC-PK1-FBPase+ monolayers and on
epithelia grown on permeable tissue culture inserts. Cultured renal
epithelia differentiate more when grown on a microporous support
because nutrients, hormones, and other factors readily gain access to
the basal surface of the epithelium (14, 17, 20, 48). This is well
documented in Fig. 6. LLC-PK1-FBPase+ cells
grown on porous supports form an epithelial layer of differentiated cells with columnar appearance and a significantly higher cell density
compared with monolayer cultures grown on plastic.
LLC-PK1-FBPase+ epithelia exhibit a
transepithelial apical negative potential difference of
1.5 mV
and a transepithelial resistance of ~150
/cm2 (17,
20). Furthermore, the cultured epithelium generates a transepithelial
pH gradient by apical proton secretion and is able to maintain pH
gradients that are imposed by adding acidic media on either side of the
filter insert (Fig. 7). All of these parameters are strong indicators
of the integrity, the transport activity, and the barrier function of
LLC-PK1-FBPase+ epithelia under the applied
culture conditions.
In LLC-PK1-FBPase+ cells, two primary PDG mRNAs
of 5.0 and 4.5 kb in size are present in subconfluent and confluent
cultures (38). The same RNAs could also be readily detected in the
present study in confluent dish- and filter-grown
LLC-PK1-FBPase+ epithelia (Figs. 8 and 12).
Specific detection of the two PDG mRNAs was achieved by using
separate cDNA probes (Fig. 2). The porcine cDNA probe, p2400, which is
derived from the 3'-untranslated region of pGA201,
hybridizes specifically to the 5.0-kb PDG mRNA, whereas the rat
and porcine cDNA probes r1500 and p930, respectively, contain
segments of the coding region of the renal PDG mRNA and hybridize to
both the 5.0- and the 4.5-kb mRNAs. The homologous porcine p930 cDNA
probe produced stronger signals than the rat r1500 cDNA.
The 5.0- and 4.5-kb PDG mRNAs differ substantially in their response to
alterations of the extracellular medium. Only the levels of the 4.5-kb
PDG mRNA are increased when LLC-PK1-FBPase+
cultures are incubated with acidic (Fig. 8) or low-potassium-containing media (Fig. 12). The 5.0-kb mRNA species appears to be constitutively expressed because its cellular levels were unaltered under all experimental conditions tested. Although the 5.0- and the 4.5-kb PDG
mRNAs share an identical stretch of coding sequence (Fig. 2), they
clearly contain different 3'-untranslated regions (38). The
3'-untranslated region of the 5.0-kb porcine PDG mRNA lacks the
eight-base, AU-rich sequence that was identified as the rat PDG mRNA
pH-response element (21, 30, 38). A 48-kDa cytosolic protein from rat
kidney cortex binds specifically to the AU repeats and thereby mediates
the pH-responsive stabilization of the PDG mRNA (30).
LLC-PK1-FBPase+ cells also contain a protein
that binds specifically to this sequence (O. Laterza and N. P. Curthoys, unpublished data). Thus, from the data presented here, one
would predict that the 3'-untranslated region of the 4.5-kb PDG
mRNA probably contains a pH-response element that has a sequence that
is highly homologous to that of the rat PDG mRNA. The essential
function of AU-rich elements in the 3'-untranslated regions of
eukaryotic mRNAs and their importance in mRNA stability and turnover
have been emphasized in recent reviews (6, 8, 39).
The apparent half-life of the 4.5-kb PDG mRNA is increased 2.3-fold
when the LLC-PK1-FBPase+ cells were transferred
to acidic medium (Fig. 10). This is consistent with the observed 2.5- to 3-fold increase in this mRNA. Thus the level of the 4.5-kb PDG mRNA
in LLC-PK1-FBPase+ cells is regulated by
extracellular pH in a manner identical to that established for the PDG
mRNA in rat kidney proximal tubular cells in vivo (9, 10, 24, 25, 41,
42). Furthermore, as seen in vivo (7, 22, 24, 25, 42), the adaptive
increase in the level of cytosolic PEPCK mRNA in
LLC-PK1-FBPase+ cells is mediated by an
increased rate of transcription and not by changes in the rate of
turnover of the PEPCK mRNA (23, 31).
By culturing LLC-PK1-FBPase+ epithelia on
microporous filter inserts, the potential sidedness of the signal that
initiates the pH response could be studied (26). As shown in Figs. 8
and 9, both epithelial surfaces must be acidified to elicit a maximal increase in PDG and cytosolic PEPCK mRNAs. This is consistent with what
occurs in vivo during metabolic acidosis, where basolateral pH is
lowered by the acidic interstitium and apical pH is lowered by the decreased filtered load of
HCO
3 and by activation of the
Na+/H+ antiporter. However, in vitro studies of
ammonia production by using isolated perfused mouse proximal tubule
segments produced different results (33, 34). When the peritubular pH
was acutely lowered by decreasing the
HCO
3 concentration of the bath buffer,
ammonia production increased by 50% (34). However, no increase in
ammonia production was observed in the perfused proximal
tubules when only the luminal perfusion pH was lowered (34). The
different effects observed with isolated perfused proximal tubules and
cultured renal epithelia may reflect a difference in how the
intracellular pH is affected in the two systems. Alpern and Chambers
(3) have clearly demonstrated that with isolated perfused segments a
reduction in basolateral pH produces a greater fall in
intracellular pH than the same reduction in luminal pH. In
contrast, studies with LLC-PK1 cells grown in plastic
dishes and on microporous inserts have shown that changes in
extracellular pH produce similar changes in intracellular pH
over the range of pH 6.8 to 7.6 (28, 40). Therefore, the
cumulative data suggest that the pH-responsive inductions of
the PDG and PEPCK genes are not initiated by an asymmetrically
distributed membrane receptor that senses changes in
the extracellular concentration of either H+ or
HCO
3 ions. Instead, the observed
responses are likely to be initiated in response to a decrease in
intracellular pH.
To further test this hypothesis, experimental conditions were selected
where the intracellular pH is decreased while normal extracellular pH
is maintained. This was accomplished by adapting cultured cells to
low-potassium-containing media (Figs. 11 and 12). Potassium depletion
leads to cell acidification (1). A low intracellular pH would enhance
luminal H+ secretion through activation of the luminal
Na+/H+ exchanger and cause an enhanced
HCO
3 reabsorption (5). Indeed, chronic
hypokalemia does increase the activity of the renal proximal tubule
apical membrane Na+/H+ exchanger, encoded by
NHE3 (2, 4). This response was recently reproduced in an in vitro cell
culture system (5). Thus the observations in the present study that the
low-potassium medium increases the rates of ammonia production (Fig.
11) and produces an increase in both the 4.5-kb PDG and cytosolic PEPCK
mRNAs (Figs. 12 and 13) that closely approximate the responses observed
with acidic medium strongly support the hypothesis that enhanced
ammoniagenesis and increased expression of the two gene products are
initiated by a decrease in intracellular pH.
In summary, LLC-PK1-FBPase+ cells, a
gluconeogenic renal epithelial cell strain, respond to acidic medium
(pH 6.9, 9 mM HCO
3) with an increase
in transcription of the cytosolic PEPCK mRNA and a pronounced
stabilization of the 4.5-kb PDG mRNA. The inability of the parental
LLC-PK1 cells to exhibit the latter response could be due
to a variety of reasons. For example, LLC-PK1 cells may not
express the mRNA-binding protein or they may lack the necessary signaling mechanism that senses changes in intracellular pH and initiates the enhanced interaction necessary to stabilize this variant
of the PDG mRNA. The observation that the in vivo response to metabolic
acidosis is reproduced in LLC-PK1-FBPase+
cultures in vitro strongly indicates that increased expression of the
two gene products is not mediated by a circulating humoral factor. When
LLC-PK1-FBPase+ epithelia are grown on
permeable filter inserts, both the apical and the basolateral sides
must be acidified to elicit the full adaptive response. This
observation and the finding that low-potassium medium elicits an
identical response suggest that the adaptive response is initiated by a
decrease in intracellular pH. Therefore, the renal cells must possess a
biochemical mechanism for directly sensing changes in intracellular pH
and a pathway to transduce this information into a signal that alters
the expression of the two enzymes. Thus the
LLC-PK1-FBPase+ strain is a pH-responsive
permanent renal cell line that should prove valuable as a tissue
culture model to further characterize how renal proximal tubular cells
sense pH and how this signal is transduced to increase nuclear
transcription and cytosolic mRNA stabilization of specific gene
products during metabolic acidosis (12).
 |
ACKNOWLEDGEMENTS |
The present study was initiated during a research semester of G. Gstraunthaler at Colorado State University. This work was supported by
the Austrian Science Foundation, Grants P11126 and P12705 (to G. Gstraunthaler) and by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-37124 (to N. P. Curthoys).
 |
FOOTNOTES |
Parts of this study were presented at the Annual Meetings of the
American Society of Nephrology in Orlando, FL, 1994 (J. Am. Soc.
Nephrol. 5: 304, 1994) and San Antonio, TX, 1997 (J. Am. Soc.
Nephrol. 8: 51A, 1997).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Gstraunthaler, Institute of Physiology, Univ. of Innsbruck,
Fritz-Pregl-Str. 3, A-6010 Innsbruck, Austria (E-mail:
gerhard.gstraunthaler{at}uibk.ac.at).
Received 24 February 1999; accepted in final form 12 October 1999.
 |
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