Glutamate transport asymmetry in renal glutamine
metabolism
Patsy
Carter and
Tomas C.
Welbourne
Department of Cellular and Molecular Physiology, Louisiana State
University Medical Center, Shreveport, Louisiana 71130
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ABSTRACT |
D-Glutamate (Glu) was
previously shown to block L-Glu
uptake and accelerate glutaminase flux in cultured kidney cells
[Welbourne, T. C., and D. Chevalier. Am. J. Physiol. 272 (Endocrinol.
Metab. 35): E367-E370, 1997]. To test
whether D-Glu would be taken up by the intact functioning kidney and effect the same response in vivo,
male Sprague-Dawley rats were infused with
D-Glu (2.6 µmol/min), and
renal uptake of D- and
L-Glu was determined from chemical and radiolabeled arteriovenous Glu concentration differences times renal plasma flow. The amount removed was then compared with that
amount filtered to obtain the antiluminal contribution. In the
controls, L-Glu uptake measured
as net removal was 33% of the arterial
L-Glu load and not different
from that filtered, 27%; however, the unidirectional uptake was
actually 58% of the arterial load, indicating that antiluminal uptake
contributes at least half to the overall Glu consumption. Surprisingly,
the kidneys showed a more avid removal of
D-Glu, removing 73% of the arterial load, indicating uptake predominantly across the antiluminal cell surface. Furthermore, uptake of
D-Glu was associated with a 55%
reduction in L-Glu uptake, with
the residual amount taken up equivalent to that filtered;
D-Glu did not increase the
excretion of the L-isomer.
However, elevating plasma L-Glu
concentration reduced uptake of the
D-isomer, suggesting a shared
antiluminal transporter. Thus there is an apparent asymmetrical
distribution of the D-Glu
transporter. Under these conditions, kidney cortex L-Glu content decreased 44%,
whereas net glutamine (Gln) uptake increased sevenfold (170 ± 89 to
1,311 ± 219 nmol/min, P < 0.01) and unidirectional uptake nearly threefold (393 ± 121 to 1,168 ± 161 nmol/min, P < 0.05); this
large Gln consumption was paralleled by an increase in ammonium
production so that the ratio of production to consumption approaches 2, consistent with accelerated Gln deamidation and subsequent Glu
deamination. These results point to a functional asymmetry (antiluminal
vs. luminal) for Glu transporter activity, which potentially plays an
important role in modulating Gln metabolism and renal function.
glutamate isomers; metabolic regulation; cellular acidosis; L-glutamate and
L-[14C]glutamine
uptake; antiluminal glutamate uptake
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INTRODUCTION |
D-GLUTAMATE enhances glutamine utilization and ammonium
and alanine production in cultured kidney cells by blocking
L-glutamate uptake and
decreasing cellular L-glutamate
levels (27). Because glutamine is a major fuel for most cells (14, 25)
as well as a source of metabolically generated base in the kidneys (4), regulation of this pathway is of considerable interest. In this regard,
L-glutamate has long been
recognized as a competitive inhibitor of the phosphate-dependent
glutaminase reaction (8, 11, 23), the first committed step in the
intracellular pathway for glutamine utilization as a fuel. In
view of the cultured kidney cell study, we wondered whether
D-glutamate might also block
L-glutamate uptake and
activate the glutaminase pathway in vivo, thereby presenting a strategy
for metabolic regulation amenable to therapeutic manipulation. Although
D-glutamate is considered an
"unnatural" amino acid, it is in fact the major
D-amino acid for intestinal
bacteria and when administered in vivo was readily converted to
D-5-oxoproline (15, 19), a
finding that raises the question as to how the D-glutamate enters cells. Indeed
most (7, 24) but not all (13, 20) studies have concluded that the
D-isomer is not transported by
the acidic amino acid transporter. Recently, however, molecular cloning
and functional expression of the excitatory amino acid transporters,
EAAT-1 and EAAT-3 (2), have shown transport of the
D-isomer by both of these
subtypes, although at a lower affinity than that exhibited for the
L-isomer; note that the
D-isomer can apparently be
transported at, or even above, the maximal rate of the
L-isomer by EAAT-1. In line with
transport of D-glutamate in
vivo, Samarzija and Fromter (20) monitored renal tubular cell
depolarization as an index of transport and observed that D-glutamate was more effective
in depolarizing the cells than the
L-glutamate at millimolar
concentrations. Although it is unclear as to which cell surface was
effecting the D-glutamate
transport, these findings are consistent with
D-glutamate uptake by the intact kidney and raise the possibility of competition with the
L-isomer and consequently
activation of the glutaminase pathway.
Transporter-mediated cotransport of acid might also modulate glutamine
utilization apart from the direct glutamate effect on glutaminase
activity. Indeed, expression of the cloned rabbit glutamate
transporter, EAAC-1, in frog oocytes resulted in a fall in
intracellular pH (9), suggesting that glutamate transport, be it the
D- or
L-isomer, might effect a
cellular acidosis. Because cellular acidosis accelerates glutamate
utilization and enhances ammoniagenesis (16, 18),
D-glutamate's mode of action
could be by blocking L-glutamate
uptake and/or by delivering an acid load.
In light of the potential roles that glutamate transport plays in
modulating in vivo glutamine metabolism, our purpose was twofold:
first, to determine the extent of
D-glutamate uptake by the intact
kidney and the effect on uptake and cellular level of the
L-isomer and, second, to
demonstrate that renal glutaminase flux and ammoniagenesis are
accelerated as a consequence. The results to follow show a surprisingly
avid uptake of the D-glutamate at the antiluminal cell border leading to a fall in cellular
L-glutamate and enhanced
glutamine utilization and ammonium formation.
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MATERIALS AND METHODS |
All experiments were performed on adult male Sprague-Dawley rats
(Harlan, Indianapolis, IN) weighing between 350 and 450 g. Animals were
housed one per cage and maintained on Purina Rat Chow. Food, but not
water, was removed 16 h before an experiment. The animals were
anesthetized with Inactin (120 mg/kg) and maintained at 37°C after
insertion of a tracheal cannula and were prepared for renal extraction
studies as previously described (5). After reaching equilibration,
animals were either continuously infused for an additional 30 min with
saline (time control) or infused with
D-glutamate (neutralized with
sodium bicarbonate) added to the saline vehicle and infused at the rate
of 2.6 µmol/min after a 50-µmol
D-glutamate prime injection.
Urine was collected under oil from an indwelling bladder catheter over
two consecutive 15-min periods; 0.3-ml blood samples were
simultaneously drawn from the carotid artery and left renal vein at the
end of each period with blood replacement from a donor littermate.
Immediately after these, ~200-mg sections of the outer kidney cortex
were cut and promptly homogenized in ice-cold 5% TCA for kidney cortex
amino acid analysis (left kidney) and in 0.44 M sucrose, 10 mM
MgCl2, and 0.1 M Tris buffer (pH
7.2) using a polytron (half-speed for 30 s) for the ammoniagenesis from
D-glutamate assay (right
kidney).
For
L-[14C]glutamate
and
L-[14C]glutamine
unidirectional uptake studies, the carotid artery cannula was moved
down to the junction with the aorta for direct infusion of
L-[U-14C]glutamate
(158 mCi/mmol, Sigma, St. Louis, MO) or
L-[14C]glutamine
(253 mCi/mmol, NEN, Boston, MA) into the aorta (5); [14C]glutamate was
given as a priming dose, 4 µCi at 45 min followed immediately by the
constant infusion at the rate of 1 µCi/min over 30 min;
L-[14C]glutamine
was infused at twice this rate. Arterial blood samples were drawn from
the cannulated right femoral artery.
Experimental design.
To discern an in vivo effect of
D-glutamate on
L-glutamate uptake into the
kidney, two approaches were used. Removal of
L-glutamate by the kidneys was
determined from the arteriovenous glutamate concentration difference
times renal plasma flow; this of course measures net removal, the
summation of unidirectional uptake and simultaneous release. Renal
plasma flow was estimated from creatinine clearance divided by the
fraction of arterial creatinine removed by the kidneys as previously
described (5). Arterial plasma and filtered glutamate loads were
calculated from the renal plasma flow and glomerular filtration rate
times arterial plasma concentration as previously described (5);
fractional extraction was calculated as the net uptake divided by
arterial load. Fractional reabsorption was calculated from the amount
reabsorbed divided by the amount filtered. Net uptake of both glutamate
and glutamine was corrected for excretory loss. To measure the
unidirectional glutamate uptake, we used the
L-[14C]glutamate
tracer and fractional removal (arteriovenous
L-[14C]glutamate
difference divided by the arterial
L-[14C]glutamate)
times the arterial glutamate load to give unidirectional uptake; this
of course only measures the unidirectional uptake of
L-glutamate without the
confounding simultaneous release. Tracer amounts of
[14C]glutamate were
infused to constant specific activity (arterial sp act 142 ± 14 and
153 ± 23 cpm/nmol at 15 and 30 min, respectively); arterial plasma
glutamine specific activity was <10% of that for glutamate. We took
advantage of the enzymatic determination of L-glutamate using glutamate
dehydrogenase (27) to measure the effect of
D-glutamate (determined as total
glutamate analyzed by HPLC minus
L-glutamate) on simultaneous
L-glutamate uptake. Thus this
design provided two independent measures of the
D-glutamate effect on
L-glutamate uptake:
1) chemical, expressed as net
removal, and 2) isotopic, expressed
as unidirectional uptake. As indexes of glutaminase flux, we used three
independent measures (5): net glutamine removal, total ammonium
production (combined renal venous + urine ammonium), and unidirectional
glutamine uptake. Radiolabeled glutamine administered as a prime (4 µCi) followed by a constant infusion (2 µCi/min) via the aorta
achieved a constant arterial plasma specific activity at 15 and 30 min
(137 ± 25 and 149 ± 43 cpm/nmol at 15 and 30 min,
respectively), with the arterial radiolabeled glutamine in equilibrium
with the kidney cortex glutamine determined on the TCA extracts of
outer cortex described above as judged by their similar specific
activities measured at 30 min (149 ± 43 and 184 ± 43 cpm/nmol,
respectively). We used the ratio of ammonium produced to glutamine
utilized of 2 as an index for complete deamination and deamidation of
the extracted glutamine during the
D-glutamate infusion. Note that
D-glutamate does not support
ammonium production in rat kidney homogenates as measured by ammonium
formed in the presence of 5 mM
D-glutamate vs. that formed
in the absence of exogenous substrates 150 mM
KH2PO4
buffer (pH 7.2 alone; 1.89 ± 0.19 vs. 2.13 ± 0.04 nmol · min
1 · mg
protein
1, respectively;
n = 3).
Analyses.
Plasma, blood, and urine concentrations of creatinine, ammonium,
glutamine, and glutamate were analyzed as described (5). Glutamine and
glutamate were determined in TCA extracts of plasma and kidney by HPLC
after precolumn derivatization with
o-phthalaldehyde (OPA). Radiolabeled
glutamate and glutamine OPA derivatives (retention times 7.3 and 11.4 min, respectively) were collected under their respective peaks and
detected by liquid scintillation spectrometry as described (5, 16).
Recovery of stock radiolabeled
L-[14C]glutamate
and
L-[14C]glutamine
OPA derivatives added to the column and isolated under the respective
peaks was 95 ± 6 and 87 ± 5%, respectively
(n = 3). Homoserine (1 mM) was
routinely added to the TCA extract as an internal standard. To measure
L-glutamate concentration in the D-glutamate infusion
studies, the previously described fluorometric-enzymatic assay was
employed (27). The D-glutamate
concentration was taken as the difference between total glutamate
determined by HPLC and the
L-glutamate determined by
enzymatic assay. Recovery of
L-glutamate from an equimolar
(0.125 mM) mixture of L- and
D-glutamate was 103 ± 1% (n = 3); although
D-glutamate (Sigma) is listed as
99% pure, some 6 ± 2% of the
D-glutamate reacted as the
L-isomer in this assay, thus
making the actual recovery closer to 97%. The HPLC-determined values
for total glutamate in the absence of infused D-glutamate were assumed to
represent L-glutamate, although
there was in fact a 14 ± 4% discrepancy between the HPLC and
enzymatic analysis of arterial plasma glutamate, suggesting the
presence of the D-isomer. In
contrast, L-glutamate standards
gave excellent agreement between the two methods (HPLC = 102 ± 5%
of enzymatic assay, n = 6, at 1 mM
L-glutamate). Nevertheless, we
assumed that, in the time-control group, glutamate represented solely
the L-isomer and was therefore
measured by HPLC.
Statistical comparisons between time-control and
D-glutamate-infused groups
were made with the Student's t-test
and either a one-tailed or two-tailed
t table depending on whether an a
priori directional change was postulated (see introduction).
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RESULTS |
Figure 1 shows the arterial glutamate load
delivered compared with that extracted by both kidneys for the control
and D-glutamate-infused groups
(Table 1 presents these and other pertinent
data). Infusing D-glutamate at
the rate of 2.6 µmol/min resulted in an arterial plasma
D-glutamate concentration of 242 ± 25 nmol/ml vs. 142 ± 9 nmol
L-glutamate/ml for time control
(Table 1) and a D-glutamate load
to the kidney some 2.4-fold greater than time controls (2,581 ± 217 nmol D-glutamate/min vs. 1,075 ± 131 nmol L-glutamate/min, respectively) due to rise in both the arterial plasma
D-glutamate concentration and
renal plasma flow with
D-glutamate infusion (Table 1).
Although the control group's kidneys removed a respectable 33% of
their arterial L-glutamate load,
surprisingly those infused with
D-glutamate removed an even
greater percentage, 73%, of the arterial
D-glutamate load (Table 1). This
highly efficient extraction rivals that observed for substances, such
as p-aminohippurate (PAH), which are
taken up from the peritubular capillaries. Infusing D-glutamate depressed the
simultaneously measured
L-glutamate removal from 33 to
24% of that delivered (355 ± 38 vs. 224 ± 53 nmol/min for time
controls and D-glutamate,
respectively, P < 0.05; Table 1).
This decrease was associated with a fall in the arterial
L-glutamate concentration (142 ± 9 to 83 ± 12 nmol/ml, P < 0.05; Table 1) as well as a reduction in the arteriovenous L-glutamate concentration
difference (48 ± 5 to 22 ± 6 nmol/ml, P < 0.05; Table 1). Thus the fall in
extraction was associated with a lowered arterial plasma
L-glutamate concentration while the arterial load was maintained (Fig. 1) by the enhanced renal plasma
flow (Table 1). When the glutamate removed from the plasma was compared
with the amount filtered at the glomerulus, the results become even
more revealing. With the
D-glutamate, the kidneys removed
an amount of D-glutamate from
the blood that exceeded the amount filtered by 132% (Fig. 1; 1,882 ± 227 vs. 813 ± 93 nmol/min for removal and
filtration, respectively; Table 1); in the time control,
L-glutamate removed exceeded the
amount filtered by only 6% (355 ± 38 vs. 334 ± 35 nmol/min,
respectively; Table 1). These results are consistent with
D-glutamate being transported into the kidneys predominantly across the antiluminal cell surface (blood side), whereas clear-cut antiluminal
L-glutamate uptake is not
apparent.

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Fig. 1.
Renal glutamate (Glu) handling in control and
D-Glu-infused groups. Arterial
plasma load delivered to kidneys and that filtered across glomerulus
were calculated from renal plasma flow and glomerular filtration rate
(GFR) times arterial plasma concentration, whereas amount removed was
determined from arterial minus renal venous Glu concentration
difference times renal plasma flow. Results are means ± SE; see
Table 1 for additional pertinent data. * Difference in
L-Glu removal between time
control and D-Glu infused is
significant at P < 0.05.
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However, L-glutamate transport
into the kidney cells across the antiluminal cell surface can in fact
be demonstrated from the unidirectional uptake as shown in Fig.
2 and Table
2. From the fractional extraction of
L-[U-14C]glutamate
and the arterial L-glutamate
load, one obtains the unidirectional transport of
L-glutamate into the kidney (see
MATERIALS AND METHODS,
Experimental design for details); this
effectively eliminates the confounding factor of the simultaneous
release of "cold"
L-glutamate, which is included
when net amounts removed are determined (arteriovenous concentration
difference times renal plasma flow, see MATERIALS AND
METHODS, Experimental
design). As depicted in Fig. 2 and in detail in Table
2, the unidirectional L-glutamate uptake into the
kidney was nearly double the amount that was filtered across the
glomerulus (544 ± 12 vs. 283 ± 46 nmol/min,
P < 0.01) and much higher than that
detected by measuring net removal (544 ± 12 vs. 297 ± 10 nmol/min, P < 0.05; Table 2). Therefore almost half of the
L-glutamate normally delivered
to the kidneys is taken up at the antiluminal cell surface. With D-glutamate infusion, the
unidirectional L-glutamate
uptake is reduced 55% (544 ± 12 to 245 ± 66 nmol/min,
P < 0.02) to a value that is not
different from the amount filtered at the glomerulus (Fig. 2).
D-Glutamate infusion did not
increase L-glutamate excretion as shown in Table 1, whereas luminal
L-glutamate uptake was more avid
than the D-glutamate uptake
(99.7 ± 0.1 vs. 97.7 ± 0.7% fractional reabsorption,
respectively) in contrast to the fractional extraction (25 ± 5 vs. 73 ± 9% fractional extraction, respectively, Table 1). These findings are consistent with the studies showing that the
D-isomer does not compete with
L-glutamate for transport into the cell at the luminal cell surface (6, 24).

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Fig. 2.
Unidirectional Glu uptake compared with amount filtered in control and
D-Glu-infused groups.
L-[U-14C]Glu
was infused to a constant specific activity and uptake determined as
described in MATERIALS AND METHODS;
filtered Glu was determined from arterial Glu concentration times GFR.
Results are means ± SE from n = 4;
see Table 2 for pertinent data. * Difference between
D-Glu infused and control uptake
significant at P < 0.05.
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The fall in arterial L-glutamate
concentration during D-glutamate
infusion (Table 1) raises the possibility of an effect of
D-glutamate on some extrarenal
site, and this might cause the renal uptake to fall. Therefore
L-glutamate was also infused, raising the L-isomer to
concentrations well above the time-control level. As shown in Fig.
3, when the arterial plasma ratio of
D- to
L-glutamate increases despite an
elevated L-glutamate plasma concentration, the L-glutamate
uptake falls. At the lowest D- to L-glutamate ratio, 0.2 (56 and 231 nmol/ml for D- and
L-glutamate, respectively), 30 and 34% of their arterial loads were extracted; at a ratio of 0.4 (110 and 273 nmol/ml, respectively),
L-glutamate extraction dropped
to 21 as D-glutamate rose to
44%; at the ratio of 1.2 (198 and 158 nmol/ml, respectively),
L-glutamate uptake was pretty
nearly abolished with 89% of the
D-glutamate extracted. Note that
neither L- nor
D-glutamate excretion rates
contributed significantly to the uptake rates (excreted glutamate being
<1% of their respective uptake rates). Therefore these results
demonstrate that the effect of
D-glutamate on
L-glutamate uptake cannot be attributed to the spontaneous fall in arterial
L-glutamate concentration but
instead points to a competitive interaction between these isomers in
the kidney and specifically at the antiluminal surface.

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Fig. 3.
Uptake of Glu isomers at different arterial concentration ratios for
D- and
L-Glu. Relative
D-, L-Glu concentrations were
(in nmol/ml) 56/231, 100/273, 186/232, and 198/158, giving arterial
plasma ratios 0.2, 0.4, 0.8 and 1.25, respectively. Results are from 4 rats.
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The D-glutamate-induced
reduction in L-glutamate uptake
results in a fall in cortical
L-glutamate content, as shown in
Fig. 4. With
D-glutamate infusion, cortical
L-glutamate content decreased 44% (37 ± 6 to 17 ± 3 nmol/mg protein,
P < 0.01) with the appearance of
D-glutamate (29 ± 2 nmol/mg
protein). Based on some 7 µl cell water/mg protein, these values
translate to 5.3, 2.4, and 4.1 mM glutamate, respectively. The decrease
in L-glutamate is well within
the range of modulation of the glutaminase's inhibition constant
(Ki) of 5 mM
for L-glutamate (23). Also shown
are the cortical glutamine contents for the 2 groups; compared with the control, D-glutamate infusion
reduced glutamine 27%, resulting in a reduction in the
L-glutamate to
L-glutamine ratio (3.7 ± 0.9 to 1.9 ± 0.4, P < 0.05)
consistent with deinhibition of the glutaminase flux.

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Fig. 4.
Renal cortical Glu and glutamine (Gln) content in sections of cortex
obtained at end of time control and
D-Glu infusions (see
MATERIALS AND METHODS). Results are
means ± SE from n = 13 time
controls and 8 D-Glu-infused rat
kidneys per group, respectively. * Difference significant from
time controls at P < 0.05.
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The effects of D-glutamate on
glutamine utilization and ammonium production are presented in Fig.
5 and Tables 3
and 4. In the control groups, net glutamine uptake
was small and much less than the total ammonium production rate (Fig.
5, Table 3), indicating that other ammoniagenic precursors are
supporting the control ammonium production rate, as previously noted
(4, 5). With D-glutamate, there
is a sevenfold elevation in glutamine removal (170 ± 89 to 1,311 ± 219 nmol/min, P < 0.01),
whereas ammonium production rises to a ratio (2,433 ± 326 /
1,311 ± 219 nmol/min) consistent with complete deamidation and
deamination of the consumed glutamine. Note that although glutamine
excretion increased with
D-glutamate (2.5 ± 0.3 to
4.1 ± 0.7 nmol/min, P < 0.05), this would not contribute significantly to the increased net
uptake. To confirm the accelerated glutaminase flux, unidirectional glutamine uptake rates were measured from the fractional extraction of
[14C]glutamine. As
shown in Table 4, glutamine's unidirectional glutamine uptake rate
increased nearly threefold (393 ± 121 to 1,168 ± 161 nmol/min,
P < 0.05) with
D-glutamate and
L-glutamate plasma
concentrations of 269 ± 33 and 51 ± 9 nmol/ml. At these arterial D- and
L-glutamate concentrations,
glutamate net uptake decreased 42% (314 ± 45 to 182 ± 27 nmol/min, P < 0.05), with a large
uptake of D-glutamate
(1,746 ± 516 nmol/min), in agreement with the results obtained in
the previous experiments and presented in Table 1. The infusion of
D-glutamate tended to reduce
glutamine production (377 ± 185 to 153 ± 90 nmol/min,
P = 0.10) so that the net glutamine
extraction would increase by 224 nmol/min. Nevertheless the accelerated
rate of glutamine breakdown (373 to 1,168 or 775 nmol/min) was a far
greater contributor to the enhanced net uptake (998
13, or 985 nmol/min) than the 224 nmol/min gained from the fall in glutamine
synthesis.

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Fig. 5.
Renal Gln consumed and ammonium (NH+4)
released by time-control and
D-Glu-infused groups measured
after 30 min of infusion. Gln consumed represents net uptake determined
from arteriovenous concentration difference times renal plasma flow
over 15- to 30-min period. NH+4 released is
sum of that released into renal vein and urine (see
MATERIALS AND METHODS for details).
Results are means ± SE; see Table 3 for pertinent data.
* Differences from time controls significant at
P < 0.05.
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D-Glutamate uptake was
associated with a fall in urinary pH (6.56 ± 0.08 to 6.07 ± 0.06 units, P < 0.01). Because
filtered bicarbonate would have increased in conjunction with the rise in glomerular filtration, the increased urinary acidification is
probably due to the movement of acid into tubule cells in association with the D-glutamate and
subsequent secretion of the proton.
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DISCUSSION |
The goals of this study were twofold:
1) to demonstrate that
D-glutamate could in fact be
extracted by the functioning kidney and
2) to confirm that blocking
L-glutamate uptake and reducing the cellular
L-glutamate would
accelerate the glutaminase flux (Fig.
6).

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Fig. 6.
D-Glu transporter asymmetry and
putative effects on renal Gln metabolism.
D-Glu blocks antiluminal
L-Glu uptake, lowering this
glutaminase inhibitor and thereby activating Gln hydrolysis
(1) and
NH+4 production
(2 and
3).
D-Glu transport-associated
tubule acid load (H+) would
further enhance NH+4 formation from Gln's
amino nitrogen. Relatively greater uptake of
D-isomer at antiluminal cell
surface (renal vein) as opposed to
L-Glu and vice versa for luminal
cell surface (urine) are consistent with Glu transporter asymmetry in
functioning organ.
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Although we suspected some
D-glutamate extraction by the
kidneys, the avidity and quantitative extent were truly remarkable. Indeed, almost 75% of the
D-glutamate delivered to these
kidneys was removed in a single pass, an uptake efficiency that is far greater than that seen with the
L-isomer or, for that matter, greater than that for
L-glutamine, a major renal fuel,
and that approaches that of PAH, which is taken up from the peritubular capillaries. This large extraction was all the more surprising in that
reabsorption of D-glutamate does
not occur in the proximal tubule (24, 26), but
D-glutamate transport does occur
in the loop segment by an ill-defined system that accepts both
L- and D-isomers and yet shows no
competitive inhibition (6). Thus the displacement from the uptake
process of the L-isomer by
D-glutamate as shown in vivo
(Fig. 3) as well as in vitro (27) is not likely to reflect competition
with transport of L-glutamate at
the luminal surface. We favor the antiluminal cell surface as the site
at which D-glutamate effects the
reduction in L-glutamate
uptake, for the following reasons: first,
significant L-glutamate
transport into the kidney cells occurs across this surface, at
least 50% of the L-isomer
extracted entering from the blood side (see Fig. 2), and second,
D-glutamate infusion
decreases the L-glutamate extraction to the amount filtered that is not subject to competitive inhibition by the D-isomer. By
blocking L-glutamate uptake at the antiluminal cell surface,
D-glutamate could effect the
measured fall in cortical
L-glutamate content (Fig. 4)
without inhibiting luminal
L-glutamate uptake. This of
course implies that uptake across both cell surfaces is important to
maintain cellular glutamate concentration as proposed (20, 21).
Virtually all in vitro studies of the acidic amino acid transporter
activity have reported that
D-glutamate exhibits much less
reactivity with the transporter than does the
L-isomer of glutamate.
Paradoxically, the present in vivo study suggests that whatever
transporter activity is responsible for
D-glutamate uptake, its
reactivity toward D-glutamate in
the functioning kidney is at least as great as that for the
L-glutamate. Although the
antiluminal transporter is undefined, the apparent competitive
interaction with L-glutamate
(Fig. 3) suggests that
D-glutamate uptake may indeed
reflect the activity of some acidic amino acid transporter. If so, the
physiological conditions imposed may play a greater role in the
transporter's specificity characteristics than we previously suspected
(but see Refs. 13, 20). Besides basolateral glutamate transporters,
organic ion transporters, i.e., for PAH or lactate, are also expressed
in this membrane which could contribute to both
L- and
D-glutamate uptake. At present,
studies are being undertaken to identify and further characterize the
transporter subtype involved, i.e., EAAT-1, EAAT-2, and so forth (2).
Although D-glutamate is
generally considered an unnatural amino acid, it is probably generated
daily in considerable amounts from intestinal bacterial flora (where it
is a major component of cell walls) and found in a number of tissues,
at least in the rat (10). The present study shows a surprisingly avid
removal of the exogenously infused
D-glutamate by the kidneys (Fig.
1). Indeed, this rate of removal (~2 µmol/min) accounts for the
major fraction of the D-glutamate infused
(2.6 µmol/min), whereas the rate of
D-glutamate excretion is in fact
insignificant (0.02 µmol/min), raising the question as to its
metabolic fate. Although we did not attempt to follow its quantitative
conversion to D-5-oxoproline as
previously demonstrated (15, 19), we did confirm that
D-glutamate is not a source of
ammonium, since in neither the previous study (27) nor the present
study was ammonium production increased above that produced in the
absence of exogenous substrate. This would also be expected from
Ratner's much earlier study (19), showing that when
15N-labeled
D-glutamate was
administered to rats it was recovered in the urine as
D-5-oxoproline with virtually an
identical 15N enrichment.
According to Sekura et al. (22), this conversion of
D-glutamate to
D-5-oxoproline catalyzed by
-glutamylcyclotransferase is the major, if not only, metabolic
pathway participating in its disposal. As pointed out (22), rapid
metabolic conversion prevents intracellular accumulation of the
D-isomer, which is a potent
inhibitor of glutathione synthesis. Indeed,
-glutamylcysteine synthase exhibits a greater affinity for
D-glutamate than for the
L-isomer, effectively blocking
glutathione synthesis (22). Consequently any buildup of
D-glutamate could have effects
on a host of physiological processes dependent on glutathione and glutamine homeostasis. In the present experiments endogenous
D-glutamate appears to be
present in rat plasma, consistent with reports of D-glutamate present in
tissues (10). If so and given a
Ki of 0.8 mM for
D-glutamate in
-glutamylcysteine synthesis (22), the potential for impaired
glutathione synthesis certainly exists. It is noteworthy that we have
observed an apparent increase in circulating endogenous
D-glutamate in aged (24 mo) as
opposed to young rats (6 mo) that appears related to diminished renal function in the former. From these findings and those of the present study, one might predict an impaired glutathione synthesis and fall in
this important antioxidant with the aging process, as has in
fact been reported (12).
There is no doubt that
D-glutamate infusion results in
a large increase in the glutaminase flux and ammonium release (see Fig.
6). There are three lines of evidence that support this: first, the
sevenfold increase in net glutamine removal (Fig. 6, 1); second, the large increase in
ammonium production (Fig. 6, 2 and
3); and third, the threefold
increase in unidirectional glutamine uptake (Table 4). In addition to
the activated glutaminase flux, a decrease in glutamine synthesis also
contributes to the D-glutamate-enhanced net
glutamine uptake, although clearly less significantly than the former.
Although
-glutamyltransferase (
-GT) is present at
both the apical and basal poles of proximal tubules and converts
extracellular glutamine to glutamate and ammonium,
D-glutamate, unlike PAH, does
not activate this enzyme (26). Nevertheless the net glutamate release
observed in Fig. 3 may reflect
-GT-catalyzed extracellular glutamine
hydrolysis and the effect of
D-glutamate to block subsequent
L-glutamate uptake.
Mechanistically, activation of the intracellular glutaminase flux is
likely to result from the reduction in
L-glutamate uptake and lowered
glutamate concentration (8), since cortical glutamate content dropped
nearly 60% (Fig. 4). Activation of the normally suppressed glutaminase
is also consistent with the fall in cortical glutamine content.
D-Glutamate may also affect
ammoniagenesis by accelerating glutamate flux through glutamate
dehydrogenase as a consequence of reducing the cellular pH. Because
glutamate transport is associated with a fall in cellular pH (9), the
pH-sensitive glutamate dehydrogenase flux would be expected to increase
(18). In support of this, there is an accelerated glutamate deamination
that keeps pace with glutaminase flux, since the ammonium
produced-to-glutamine consumed ratio is pretty nearly 2. Note that the
glutaminase flux, on the other hand, is if anything decreased by a
moderate fall in cellular pH (18), so that the reduced inhibitor
concentration (L-glutamate)
constitutes the major driving force for accelerated glutaminase flux.
In addition to the increased glutamate dehydrogenase flux, there is
additional support for
D-glutamate uptake delivering an
acid load, namely, the drop in urinary pH. Because this occurs at a
time when filtered bicarbonate is increased, a fall in urine pH
indicates enhanced tubular H+
secretion, and this appears to be associated with
D-glutamate uptake
rather than the increase in glutamine uptake because the proton
contribution from glutamine enters the tubule lumen as ammonium (17)
and therefore would not contribute to the reabsorption of filtered
bicarbonate. If supported by further experimentation, these
observations provide the basis for a dual role for antiluminal D-glutamate uptake in urinary
acidification (directly) and ammoniagenesis (via glutamine metabolism).
Besides effecting urinary acidification,
D-glutamate uptake might also
contribute indirectly to the enhanced renal plasma flow actually
observed. If the apical
Na+/H+
exchanger activity present in the proximal and loop segments were
accelerated and led to enhance
Na+-HCO
3
reabsorption (1), then the reduced solute load to the load-sensing
macula densa might effect a relaxing of the afferent arteriole
"tone," resulting in increased renal plasma flow including an
elevated filtration rate. Whether this or other potential mechanisms,
i.e., local vasoactive factors, account for the increase in flow
remains to be determined.
Finally, these studies point to a potentially important role for
glutamate transport in metabolic and perhaps functional regulation as
the result of delivering an acid load at the organ sites participating in interorgan glutamate fluxes (3). Note that both the interorgan fluxes (30) as well as organ-specific transporters are under the
influence of growth factors (29) and glucocorticoid hormone (5).
Obviously the integrative role of these two fluxes, interorgan and
cellular transport, conveys considerable importance for both acid-base
and nitrogen homeostasis.
 |
ACKNOWLEDGEMENTS |
We thank Dawn Powell for superb secretarial assistance and the
Biomedical Research Foundation of Northwest Louisiana and Dr. Neil
Granger, Chairman, Molecular and Cellular Physiology Dept., for their
support.
 |
FOOTNOTES |
Address for reprint requests: T. C. Welbourne, Dept. of Cellular and
Molecular Physiology, LSUMC, PO Box 33932, Shreveport, LA
71130-3932.
Received 17 November 1997; accepted in final form 28 January 1998.
 |
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