Department of Cellular and Molecular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130
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ABSTRACT |
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Renal glutamate extraction in vivo shows a
preference for the uptake of
D-glutamate on the antiluminal
and L-glutamate on the luminal
tubule surface. To characterize this functional asymmetry, we isolated
rat kidneys and perfused them with an artificial plasma solution
containing either D- or
L-glutamate alone or in
combination with the system XAG
specific transport inhibitor, D-aspartate. To confirm that
removal of glutamate represented transport into tubule cells, we
monitored products formed as the result of intracellular metabolism and
related these to the uptake process. Perfusion with
D-glutamate alone resulted in a
removal rate that equaled or exceeded the
L-glutamate removal rate, with uptake predominantly across the antiluminal surface;
L-glutamate uptake occurred
nearly equally across both luminal and antiluminal surfaces. Thus the
preferential uptake of
D-glutamate at the antiluminal and L-glutamate at the luminal
surface confirms the transport asymmetry observed in vivo. Equimolar
D-aspartate concentration blocked most of the antiluminal
D-glutamate uptake and a
significant portion of the luminal
L-glutamate uptake, consistent
with system X
AG activity at both
sites. D-Glutamate uptake was
associated with 5-oxo-D-proline
production, whereas L-glutamate uptake supported both glutamine and
5-oxo-L-proline formation; D-aspartate reduced production
of both 5-oxoproline and glutamine. The presence of system
X
AG activity on both the luminal
and antiluminal tubule surfaces, exhibiting different reactivity toward
L- and
D-glutamate suggests that
functional asymmetry may reflect two different
X
AG transporter subtypes.
glutamate isomers; D-aspartate; system
XAG subtypes; excitatory amino
acid transporter carrier 1; GLT1 glutamate transporter; 5-oxoproline; glutamine; ammonium
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INTRODUCTION |
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WE RECENTLY DEMONSTRATED IN VIVO that renal glutamate
uptake expresses a functional asymmetry, with preference for
L-glutamate at the luminal
uptake site and the D-isomer of
glutamate at the antiluminal tubule surface (4). Furthermore, the
antiluminal site was far more active in removing the
D-isomer, resulting in 70% of
the delivered D-glutamate taken
up in a single pass in contrast to 50% or less of the delivered
L-glutamate. In addition, antiluminal D-glutamate could be
reduced by increasing the
L-glutamate concentration,
suggesting that both isomers may compete for a common transport
mechanism (4). Although this antiluminal transporter has yet to be
characterized, uptake at the luminal site is consistent with the
presence of the system XAG subtype excitatory amino acid transporter carrier 1 (EAAC1) (10) along the
proximal tubule brush border and loop segments (25). More curious,
therefore, is the D-glutamate
transporting activity, because in vitro studies using various cellular
and subcellular preparations (1, 13, 26) have shown that system
X
AG readily transports the
D-isomer of aspartate but is far
less reactive with the corresponding
D-glutamate (1, 8, 26). Note
that system X
AG (8) is actually
composed of a closely related family of proteins (subtypes), all of
which transport the D-isomer of
aspartate as well as L-isomers
of glutamate and aspartate (1, 8). This suggests either that the
transporter activity responsible for the avid
D-glutamate uptake at the
antiluminal surface is not the classical
X
AG, possibly a generic organic
acid transporter, i.e., citrate transporter (6), or that in the
functioning kidney this transporter's activity is altered by the
imposed physiological conditions. Therefore, we undertook the present
study to provide more information on this antiluminal uptake process in
the context of the functioning organ.
Our second objective is to elucidate the metabolic fate of the glutamate taken up and particularly in relationship to the asymmetrical transporter expression. We know that the rat kidney expresses glutamine synthetase acivity (7, 28) and that this activity is localized to the same nephron segment [pars recta (3)] at which the luminal EAAC1 transport protein is most highly expressed (25). We also know that glutamate dehydrogenase activity is present along the proximal tubule (28), so that both ammonium and glutamine are expected products of the transported L-glutamate. D-Glutamate, on the other hand, is thought to be converted to 5-oxo-D-proline and excreted (16, 21). If so, one expects the D-glutamate uptake to be coupled to 5-oxo-D-proline formation and L-glutamate uptake to support both ammonium and glutamine production.
We chose the isolated functioning rat kidney for these studies for the
following reasons. First, in isolation, the functioning organ could be
presented with a single isomer, particularly critical for studying
D-glutamate. In vivo, of course
L-glutamate is always present,
both that preformed and delivered in plasma as glutamate and that
generated locally from
L-glutamine (32). Consequently, a study of D-glutamate uptake
alone is not possible in the in vivo functioning organ (4). However,
the isolated kidney perfused with a single isomer offers the
opportunity of providing a clear view of its removal rate as well as
the relative contribution of the luminal and antiluminal uptake sites.
Second, the isolated kidney preparation allows the monitoring of both
glutamate removal and product formation rates. Because site-specific
transporter activity is localized on opposite poles of the renal
tubules, measurement of product formation in light of the dominant
transporter site provides insight into the functional role of that
specific transporter. This approach may be particularly important in
regard to the findings in models in which system
XAG subtypes have been
"knocked-out" (20). Finally, the isolated kidney functions with a
nearly normal glomerular filtration rate and proximal tubule activity
(14), processes important for physiological fidelity in monitoring
glutamate uptake and metabolism in comparison with the intact
functioning kidney.
We therefore deployed the isolated kidney perfused with either
D- or
L- glutamate, alone or in
combination with D-aspartate, to
specifically block system XAG, and
then we determined the uptake at the luminal and antiluminal surfaces as well as the metabolic conversion. The results to follow will show
that D-glutamate is readily
taken up by the functioning kidney, predominantly at the antiluminal
site by system
X
AG. The D-glutamate transported into
the kidney is concentrated 70-fold above the plasma level and coupled
to 5-oxo-D-proline formation. L-Glutamate uptake occurs at
both luminal and antiluminal X
AG sites associated with formation of ammonium and glutamine, and, surprisingly, with significant
5-oxo-L-proline formation.
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MATERIALS AND METHODS |
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Kidneys from fed male Sprague-Dawley rats weighing between 350 and 450 g were isolated and perfused as previously described (31). Briefly,
animals were anesthetized with inactin, 120 mg/kg, and placed on a
heated animal board with core temperature maintained at 36°C. The
right jugular vein was cannulated, and a maintenance infusion of 0.150 M NaCl containing 3% mannitol was delivered at the rate of 20 µl · min1 · 100 g body weight
1 for 0.5 h,
after which the abdomen was opened and a PE-10 catheter was inserted
into the right ureter. The right renal artery was cannulated via the
superior mesenteric artery by use of a specially modified 18-gauge
needle; the right kidney was flushed in situ, rapidly cut free, and
transferred to the perfusion chamber. The perfusate was then changed
over by means of a three-way stopcock, recycling an artificial plasma
solution containing (in mM) 120 NaCl, 4.7 KCl, 1.0 CaCl2, 1.2 MgSO4, 20 NaHCO3, 5 glucose, 0.5 pyruvate,
0.3 creatinine, and 0.01 phenol red. Ficoll, approximate mol wt 70,000 (Sigma, St. Louis, MO), was added to a concentration of 5 g/l, and the
perfusate was then filtered through a 0.22-µm filter (Millipore,
Bedford, MA) and adjusted to pH 7.40 after equilibration with 95%
O2-5%
CO2. Either
D- or
L-glutamate alone or combined
with D-aspartate was added to
the perfusate to an initial concentration of 250 µM and after 15 min
was continuously infused at the rate of 600 nmol/min (0.11 ml/min).
Eighty-five milliliters of perfusion media were initially recycled
through the kidneys at a rate of 30 ml/min for 60 min with continuous aeration, 95% O2-5%
CO2. After the first 15-min
equilibration period, three consecutive 15-min clearance periods were
observed with urine collected under oil in tared tubes, with perfusate sampled (0.5 ml) at the end of each period; urine volumes were estimated by gravimetric analysis and did not exceed 25% of the glomerular filtration. Perfusate volume remained at ~90% of the initial perfusate volume after the 1-h perfusion period.
Analysis.
Perfusate and urine creatinine concentrations were determined
colorimetrically (Sigma). Ammonium in the perfusate and urine was
isolated by the microdiffusion technique and measured colorimetrically by use of the Berthelot reagents (31). Urine and perfusate pH was
determined when drawn by use of a Corning 240 pH meter and Glass
electrode. Glutamate, aspartate and glutamine concentrations in the
perfusate and urine were analyzed on samples treated with 5% ice-cold
TCA, placed on ice for 10 min, and centrifuged at 10,000 g for 10 min. Aliquots of the
protein-cleared TCA extracts were then hydrolyzed (see below) or
directly derivatized with O-phthalaldehyde (OPA) and manually
injected onto a Microsorb-C18 column precisely after 90 s of incubation at room temperature. The
amino acids were then eluted using step gradients of methanol and
sodium acetate solvents; retention times for aspartate, glutamate, glutamine, homoserine (internal standard), and alanine were 5, 7.8, 11.3, 13.3, and 16 min, respectively. Recovery of
L- and D-glutamate (Sigma
D-glutamate optical purity
[]20D =
30.2°)
standards, 0.5 mM, added to the perfusate samples from L- or
D-glutamate-infused kidneys was
103 ± 1 and 97 ± 2% (n = 3 for
each isomer). The concentration of 5-oxoproline was measured by two
independent methods; perfusate samples were analyzed by HPLC-coupled
mass spectrometry (MS), with authentic 5-oxoproline (Sigma) used as an internal standard, and indirectly by measuring glutamate formed after mild acid hydrolysis [6 N HCl at 95°C
for 2 h (30)] of perfusate and urine samples. The hydrolyzed
samples were then neutralized with 6 N NaOH, and the sample glutamate concentration was determined by HPLC as we have described. Authentic 5-oxoproline and glutamate standards, 1 and 0.5 mM, were carried through the acid hydrolysis and used to calculate the posthydrolysis glutamate concentrations; neutralized posthydrolysis samples were spiked with homoserine as an internal standard (see retention time
previously discussed). Estimated 5-oxoproline concentration measured by
the direct and indirect methods was in reasonably good agreement with
the indirect acid hydolysis method, giving consistently higher
recoveries (110 ± 5%) than the HPLC-MS method. Note, however, that
neither method distinguishes between the
D- and
L-isomers of 5-oxoproline. To
assess whether any racemization had occurred, glutamate appearing after
acid hydrolysis was assayed enzymatically using glutamate dehydrogenase
(GDH) (2); glutamate appearing after acid hydolysis of kidneys perfused
with L-glutamate was
quantitatively detectable by the enzymatic assay (92 ± 5% of
HPLC-measured glutamate), whereas glutamate formed after acid hydrolysis of kidneys perfused with
D-glutamate was not (3 ± 5%). Note that the GDH-catalyzed reaction is stereospecific (2). To
determine the extent of the nonenzymatic conversion of
D-glutamate to 5-oxoproline,
perfusate media containing 250 µM of
D-glutamate were recirculated
minus the kidney at 37°C; after 60 min, 101% was recovered (21,500 recovered / 21,290 nmol in initial perfusate).
Calculations.
Glomerular filtration rate (GFR) was estimated from the creatinine
clearance by use of the standard formula UV/P, where U and P are urine
and the midpoint perfusate creatinine concentration and V is urine
flow. The GFR (ml/min) times the midpoint perfusate glutamate
concentration equals the glutamate load delivered to the luminal uptake
site, and this load minus the amount excreted (urine glutamate
concentration times volume 15 min) equals luminal net uptake
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RESULTS |
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Figure 1 shows the perfusate
L- and
D-glutamate concentrations
measured at 15-min intervals over the 60-min perfusion time course. The
initial perfusate D-glutamate
concentration (~250 µM) was set equal to that attained in vivo
during steady-state extraction studies (4);
L-glutamate concentration in the
second group was set at a similar level for comparison. After the first 15-min equilibration period, infusion of either glutamate isomer was
started and maintained at the rate of 600 nmol/min; this rate approximates the maximal
L-glutamate removal for the in
vivo kidney and is less than that exhibited in vivo for the
D-glutamate (4). The GFR was
initially reduced during the first equilibration period for both groups
(416 ± 60 and 414 ± 50 µl/min for
L-and
D-glutamate, respectively) and
then attained in vivo levels over the subsequent three periods (862 ± 80 and 928 ± 142 µl/min; 916 ± 72 and 992 ± 148 µl/min; and 876 ± 168 and 840 ± 126 µl/min, respectively, for
L- and
D-glutamate groups). The urine
pH decreased more for the
D-glutamate group in the
equilibration and first infusion periods (6.81 ± 0.13 vs. 7.18 ± 0.08, P < 0.05, and 6.57 ± 0.09 vs. 6.79 ± 0.18, P < 0.09) and thereafter
stabilized (6.41 ± 0.18 vs. 6.52 ± 0.05 and 6.51 ± 0.14 vs. 6.63 ± 0.03, respectively). The perfusate
D-glutamate concentration
decreased over the first 15 min (Fig. 1) and continued to decline
throughout the infusion period. The perfusate
L-glutamate concentration, on
the other hand, remained unchanged over the entire 60 min of perfusion, indicating that uptake of
L-glutamate approximated the
infusion rate.
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Figure 1 also shows the effect of
D-aspartate in combination with
either glutamate isomer infusion on perfusate glutamate concentration.
D-Aspartate caused the perfusate
D-glutamate concentration to
rise, indicating inhibition of
D-glutamate uptake on the
antiluminal surface (inhibition at the luminal surface of course
reduces perfusate glutamate concentration). Similar results were
obtained when D-aspartate was
coinfused with L-glutamate (Fig.
1). Removal rates for the L- and
D-glutamate measured over the 3- to 15-min infusion periods are shown in Table
1. The removal of the
D-isomer occurred at a faster
rate than L-glutamate over the
first 15 min of infusion; the
D-glutamate removal rate then
declined, so that rates were not different over the last two periods.
When D-aspartate was coinfused
with the glutamate isomers, the removal rate of both isomers decreased
~50% in all three infusion periods. This inhibition of
D- and
L-glutamate uptake occurs at
both the antiluminal and luminal tubule borders, as shown by the
reduction in the luminal and antiluminal uptake (Figs.
2 and 3).
When L-glutamate was infused alone, luminal uptake (Fig. 2) was 42% of the total
L-glutamate removed by the
perfused kidney (240 ± 15 vs. 564 ± 24 nmol/min). In contrast, only
18% of the D-glutamate removal
could be attributed to luminal uptake (125 ± 17 vs. 699 ± 37 nmol/min). The luminal site shows a greater fractional reabsorption
rate for L- than for
D-glutamate; for
L-glutamate 97 ± 1% of
that filtered is reabsorbed (218 ± 42 / 223 ± 42 ×100) in contrast to only 84 ± 3% of the filtered
D-glutamate (125 ± 14 /
151 ± 19 ×100) despite a far higher urinary
D-glutamate concentration (108 ± 25 vs. 20 ± 5 µM for D-
and L-glutamate, respectively,
P < 0.01).
D-Aspartate reduced the luminal
uptake of both isomers (Fig. 2). Luminal
L-glutamate uptake decreased
52% (240 ± 15 to 125 ± 8 nmol/min,
P < 0.01), and
D-glutamate uptake declined 47%
(125 ± 17 to 66 ± 7 nmol/min, P < 0.01). However, in terms of fractional reabsorption,
D-aspartate was far more
effective in blocking the
D-glutamate uptake. With D-aspartate and
D-glutamate coinfused, the
fractional reabsorption dropped from 84 ± 3 to 44 ± 8%, whereas
D-aspartate fractional reabsorption was 95 ± 5%. With
D-aspartate plus
L-glutamate, the fractional
reabsorption dropped to 62 ± 9%, with a
D-aspartate fractional
reabsorption of only 59 ± 6%. Thus, with a far lower intraluminal
D-aspartate load available to
displace the D-glutamate compared with the L-glutamate (5 vs. 41%), D-glutamate
reabsorption was more effectively inhibited than the
L-glutamate. These results are
consistent with the presence of system
XAG on the luminal surface (25,
26), and specifically subtype EAAC1 (25).
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On the other hand, antiluminal uptake of
D-glutamate exceeded that of
L-glutamate (Fig. 3; 573 ± 42 vs. 336 ± 28 nmol/min, P < 0.01)
despite the lower perfusate
D-glutamate concentration (141 ± 19 vs. 279 ± 6 µM; P < 0.001, Fig. 1). The effect that D-aspartate has to block
antiluminal glutamate uptake is also consistent with system
XAG mediating uptake at this site
as well. In fact, D-aspartate
reduced antiluminal uptake of both
D- and
L-glutamate by >50% (573 ± 42 to 238 ± 27 nmol
D-glutamate/min,
P < 0.01, and 336 ± 28 to 131 ± 10 nmol L-glutamate/min,
P < 0.01). Reportedly (6), a
recently cloned dicarboxylic acid (citrate) transporter will transport
both L- and
D-glutamate, but with
exceedingly low affinity, i.e., a Michaelis-Menten constant >1 mM.
Nevertheless, because the transporter's kinetic characteristics may
differ in the functioning organ, we perfused kidneys with
D-glutamate, as above, plus 1 mM
citrate. Despite conditions expected to be highly favorable for citrate
displacement of D-glutamate
uptake, antiluminal D-glutamate
remained unchanged (747 ± 28 vs. 707 ± 52 nmol/min,
n = 3). Thus the antiluminal uptake of
the D-glutamate is about twice
that of the L-glutamate, whereas
the reverse is true for the luminal uptake site, and both these sites
express X
AG characteristics.
The metabolic fate of the glutamate taken up by the kidney was assessed
by measuring the release of glutamine, ammonium, and 5-oxoproline over
the 45-min infusion period; this is presented in Table
2. The uptake of glutamate, infused alone
or coinfused with D-aspartate,
and the release of metabolites represent averages over the three
infusion periods. Before these rates are considered, it is important to
note that the kidney contains an endogenous glutamate pool that in the
absence of exogenous L-glutamate
declines from the in vivo level of 40 ± 7 nmol/mg protein (4) to 9 ± 3 nmol/mg protein over the 1-h perfusion period. The decline of the
endogenous glutamate pool is associated with the release of ammonium,
83 ± 9 nmol/min, but not the release of glutamate, glutamine, or
5-oxoproline. However, kidneys perfused for 45 min with
L-glutamate maintained their
glutamate content at the in vivo level (48 ± 12 nmol/mg protein);
kidneys perfused with
D-glutamate had elevated kidney
glutamate content (64 ± 14 nmol/mg protein), of which 66% was in the
D-form and 34% in the
L-form (42 ± 8 and 22 ± 9 nmol/mg protein, respectively). Given 5 µl of intracellular
water per milligram of protein,
D-glutamate's intracellular
concentration achieves 8 mM, or some 70- and 90-fold higher than the
perfusate (141 µM, Fig. 1) and urine concentrations (108 µM). These
findings also suggest that the endogenous glutamate pool would not
contribute significantly to the release rates of metabolites measured
with L-glutamate infusions but
would contribute to the ammonium release rate with the
D-isomer infusion.
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The uptake of glutamate could largely be accounted for as the release of either glutamine or 5-oxoproline (Table 2). For L-glutamate, glutamine released into the perfusate would account for two-thirds of the glutamate nitrogen disappearing (372 ± 564 nmol/min × 100) if glutamate taken up serves as a precursor for the glutamine synthetase reaction located in the pars recta as well as ammonium formation catalyzed by GDH localized upstream in the proximal convoluted segment. Surprisingly, 5-oxo-L-proline released into the perfusate accounted for a significant portion of the glutamate taken up, 43% (246 ± 564 nmol/min ×100). Noteworthy, all of the 5-oxo-L-proline was found in the perfusate, with none appearing in the urine. Furthermore, this 5-oxoproline was all in the L-isomeric form, because enzymatic analysis of the acid-hydrolyzed perfusate gave essentially the same values as that measured by HPLC (92 ± 9% recovery of the HPLC-determined glutamate, n = 5). When D-aspartate was coinfused, reducing L-glutamate uptake by >50%, both glutamine and 5-oxo-L-proline fell sharply as ammonium release increased. The increase in ammonium formation suggests that either this represents ammonium that would have been incorporated into glutamine in the absence of D-aspartate or that D-aspartate accelerates the oxidative deamination of glutamate in the proximal convoluted tubule (28). In contrast to the L-isomer, infusing the D-glutamate did not support glutamine release. However, 73% of the D-glutamate disappearing (699 ± 37 nmol/min) was recovered as 5-oxo-D-proline (534 ± 56 nmol/min), of which 58% was recovered in the urine (310 ± 95 in urine out of a removal rate of 534 ± 56 nmol/min). Note that the glutamate formed after acid hydrolysis and quantitated by HPLC could not be recovered using the enzymatic assay, indicating the 5-oxoproline was of the D-isomeric form. The fact that D-glutamate uptake was largely recovered as 5-oxo-D-proline confirms that the D-glutamate was transported into kidney cells. When D-aspartate was coinfused with D-glutamate, 5-oxo-D-proline formation decreased 73% (534 ± 56 to 145 ± 15 nmol/min, P < 0.01) in association with the reduction in the antiluminal D-glutamate uptake. Note that infusing D-aspartate alone supported a significant ammonium release (158 ± 18 nmol/min), suggesting that an endogenous L-glutamate pool may be the source of this ammonium. Overall, D-aspartate infusion reduced the major glutamate metabolites more than the uptake rate, suggesting that significant accumulation of intracellular glutamate may occur under these conditions.
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DISCUSSION |
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Our first objective was to demonstrate antiluminal D-glutamate uptake in the absence of L-glutamate, as well as L-glutamate uptake in the absence of L-glutamine, both conditions impossible to achieve in vivo. The isolated kidney preparation was chosen precisely because it provided this window of opportunity and yet exhibits near normal filtration, L-glutamate reabsorption, and significant urinary acidification. Given these conditions, there was no doubt that D-glutamate is transported into tubule cells because of the concentrated uptake of D-glutamate within the kidney (70-fold higher than the perfusate concentration) and because of the 5-oxo-D-proline formation that can only be synthesized from D-glutamate inside the cell. Note that glutamate did not spontaneously form 5-oxoproline at 37°C in the absence of the kidney. Consequently, the disappearance of D-glutamate with appearance of 5-oxo-D-proline, the concentrative D-glutamate uptake, and the inhibition of this uptake by D-aspartate all support the existence of D-glutamate transport into the tubule cell. Nor was this transport of D-glutamate a minor flux compared with the L-isomer. In fact, D-glutamate's removal rate was equal to or even greater than that of the "natural" L-glutamate (Table 1). In agreement with our previous in vivo findings (4), D-glutamate removal reflected predominantly antiluminal uptake (Fig. 3), with a much smaller contribution across the luminal surface (Fig. 2). In contrast, the removal of L-glutamate reflected nearly equal uptake at the luminal and antiluminal sites (Figs. 2 and 3). When the relative contributions of these two sites to the removal of each isomer were compared, the luminal site clearly favored the L-isomer, whereas the antiluminal site played a greater role in the D-glutamate removal. Thus these findings are in line with the proposed tubular asymmetry for glutamate transporter activity based on the in vivo study (4).
There is little doubt that luminal uptake reflects the activity of the
classical XAG transporter (13, 26), as represented by the EAAC1 subtype (25), for the following reasons. First, both L-glutamate
and D-aspartate were almost
completely reabsorbed, consistent with the EAAC1's known selective
stereospecificity (8, 10, 26) and high-affinity characteristics (13).
Second, D-aspartate and
L-glutamate displaced each other
from the uptake process with nearly equal effectiveness, consistent
with competition for the transporter (13). Third,
D-glutamate exhibits a much lower fractional reabsorption, consistent with a lower affinity for the
transporter or saturation of the transporter (1, 10). In support of a
lower affinity, D-glutamate was
more readily displaced from the transporter by
D-aspartate than was the
L-glutamate. Further studies are
required to determine the number of
X
AG subtypes present as well as
the identity and role of the low-affinity transporter observed in vivo
(4).
Similarly, system XAG appears to
mediate glutamate uptake at the antiluminal site as well (Fig. 3). In
fact, equimolar D-aspartate
blocked more than one-half of D-
and L-glutamate uptake occurring
at this site. Note that
D-glutamate was taken up at the
same rate as L-glutamate at a
concentration of only one-half that of the
L-isomer, consistent with a
higher affinity for the
D-glutamate or a lower
transporter capacity at this site. The possibility that other
transporters, such as the dicarboxylic acid transporter [i.e.,
citrate transporter (6)], contribute to this antiluminal uptake
is highly unlikely on the basis of the elegant in vivo studies of
Ullrich et al. (29). In addition, in line with this, we were unable to
displace D-glutamate uptake at
this site with 1 mM citrate. Despite demonstration of the activity of
system X
AG, the identity of the
subtype that mediates L- and
D-glutamate uptake on the
antiluminal surface remains to be determined. According to Shayakul et
al. (25), EAAC1 is present only on the luminal cell surface and is
absent from the antiluminal cell surface. This raises the likely
possibility of a second subtype of the
X
AG family that may be
predominantly distributed to the antiluminal surface of the tubule
cells. In this regard, one real possibility is the GLT1 subtype, which
is highly expressed on the basolateral surface of placental basal
plasma membranes (15). Curiously, an early study of glutamate transport
across the placenta (23) used
D-glutamate as a marker for
passive diffusion despite observing that the
D-isomer moved twice as fast as
L-glutamate; the reverse was
true for the D- and
L-isomers of leucine. If
transport of the potentially toxic D-glutamate by this organ is in
fact by GLT1, it would underline the importance of the renal removal as
detailed in our findings here. In this regard we have observed that
declining renal function in the aged rat (12 mo) compared with young
rats (2 mo) resulted in an elevation in both plasma creatinine
concentration (2.45 ± 0.06 vs. 1.06 ± 0.12 mg/dl,
P < 0.05) and
D-glutamate concentration (40 ± 9 vs. 14 ± 7 nmol/ml, P < 0.05), consistent with an important role for this renal uptake process
in maintaining low plasma levels. Further studies over concentration
ranges approximating these observed in vivo are obviously necessary. In
addition, evidence for a second transporter protein in the kidney would
provide a molecular basis for the glutamate transporter asymmetry
observed in the functioning kidney.
Our second objective was to monitor the metabolic fate of the glutamate
taken up and to relate it, as far as possible, to the transporter
activities. The conversion of
D-glutamate to
5-oxo-D-proline by the
functioning kidney explains the earlier tracer study in which
D-[15N]glutamate
fed to rats was quantiitatively recovered as urinary 5-oxo-D-[15N]proline
(21). The present studies and those elsewhere (4) add the unexpectedly
dynamic renal extraction, which in the functioning kidney exceeds the
L-glutamate uptake as well as
that for L-glutamine (32), a
major metabolic fuel. The present study shows that the antiluminal
system XAG uptake is closely
coupled to D-glutamate's
metabolic conversion to
5-oxo-D-proline via
cyclotransferase (16, 30). Because this enzyme is located within the
cytosol of the cell (30), there can be no doubt that
D-glutamate removal and
5-oxo-D-proline appearance
clearly represent transport. Note also that, when recirculated in the
absence of the kidney, virtually all of the
D-glutamate was recovered at the
end of 60 min. In addition, formation of
5-oxo-D-proline could be
inhibited by D-aspartate, a
specific inhibitor of the high-affinity
X
AG transporters. Together, the
coupled D-glutamate transport
and enzymatic conversion make for a highly efficient renal clearance, accounting for almost 70% of the
D-glutamate delivered to the kidneys in vivo (4). The significance of this high renal clearance is
not well understood at present but may relate to
D-glutamate's potential
toxicity. In this regard, we know that
D-glutamate is the most potent
naturally occurring inhibitor of glutathione synthesis by virtue of
being more reactive with
-glutamylcysteine synthetase than even
L-glutamate (24). Consequently,
any buildup of D-glutamate in
plasma, such as might occur with renal disease, could well result in
reduced glutathione levels and, hence, the ability to resist oxidative
stress, as in fact has been observed (5). In this regard it may be
significant that we have observed an elevation in plasma
D-glutamate in the plasma of
cataractus patients compared with controls (47 ± 7 vs. 6 ± 2 nmol/ml, P < 0.05, unpublished observation). Whether this reflects a specific defect in the renal uptake process for D-glutamate
or a general decline in renal function remains to be determined, but a
rise in lens D-glutamate content could have clinical significance.
The uptake of L-glutamate was coupled in part to glutamine synthetase localized in the pars recta (3, 28), which coincides with the highest density of the luminal transporter EAAC1 (25). In addition, L-glutamate also supported 5-oxo-L-proline formation, as previously reported in vitro (12) and in vivo (19, 24). Nyhan and Busch (19) found that 5-oxoproline accounts for 68% of the L-glutamate taken up by the in vivo functioning kidney. Sekura et al. (24) found significant amounts of 5-oxo-L-proline after administering radiolabeled and optically pure L-glutamate in vivo. In the present study, D-aspartate blocked L-glutamate uptake and virtually eliminated 5-oxoproline formation, consistent with coupling to the high-affinity sodium-dependent transporter present at the luminal and antiluminal tubule borders. Thus our finding of a significant L-glutamate conversion to 5-oxo-L-proline may be a physiologically important metabolic pathway contributing to the steady-state glutamate concentration within the proximal tubules (22). In this regard, the relatively greater suppression of glutamine and 5-oxoproline formation with D-aspartate (perhaps affected by D-aspartate transport-mediated fall in intracellular pH) suggests that this metabolic regulation of intracellular glutamate concentration may be lost. Additional studies at physiological L- and D-glutamate concentrations are required to determine the role metabolism plays in modulating cellular glutamate concentration in the functioning kidney.
L-Glutamate uptake in the proximal convoluted tubule may be coupled to the glutamate dehydrogenase pathway localized to this nephron site (28). Formation of ammonium via this pathway and release would provide ammonium from glutamate for the downstream synthesis of glutamine at the pars recta. If so, one might expect ammonium formation to increase with suppression of glutamine synthesis in the presence of D-aspartate, as was observed. On the other hand, secretion of ammonium into the urine may segregate ammonium formed from L-glutamate from glutamine synthesis, in which case preformed ammonium (renal medulla contains up to 50 µM of ammonium) may contribute the amide nitrogen to glutamine. If so, the combination of D-aspartate and L-glutamate may increase the ammonium formation through D-aspartate's effect to lower the cell pH and drive glutamate into the mitochondria, where it can be deaminated by GDH (4, 18, 28). Note that D-aspartate is not metabolized and would not contribute to ammoniagenesis directly (8). Similarly, D-glutamate and D-aspartate increased ammonium formation, whereas neither is a substrate for either GDH or D-amino acid oxidase (24), suggesting that endogenous L-glutamate serves as the precursor of ammoniagenesis under these conditions. Obviously precise delineation of these alternative pathways requires L-[15N]glutamate studies. Nevertheless, the present findings emphasize the potential role for glutamate transporter activity in acidifying cells (11), with subsequent modulation of their metabolic (18, 28) and transport activities.
Finally, the results point to a relationship between events at the luminal tubule surface and the resulting antiluminal glutamate uptake. Specifically, the antiluminal D-glutamate uptake was greater than that of the L-glutamate over those periods when luminal acid extrusion was higher (as reflected in urinary pH). This suggests that the antiluminal glutamate transporter may be responsive to the cell pH, analogous to the luminal dipeptide transporter (27). If so, acid extrusion from the cell would raise the intracellular pH, deprotonate the transporter intracellular pH-sensitive domain, and accelerate the antiluminal D-glutamate uptake. Such a mechanism would also explain how agents that activate the apical Na+/H+ exchanger, i.e., insulin-like growth factor I (9, 33) might also enhance basal surface glutamate uptake (33). From the perspective of antiluminal uptake, modulation of transport rates would be important for growth and repair processes (33) as well as for the regulation of glutamine metabolism (28). Further studies focusing on the relationship among acid secretion, intracellular pH, and antiluminal or basal cell surface glutamate uptake are obviously necessary.
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ACKNOWLEDGEMENTS |
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We thank Tian Ma and Josh Chance for excellent technical support, and we acknowledge Genentech for funding.
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FOOTNOTES |
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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 correspondence and reprint requests: T. C. Welbourne, Dept. of Cellular and Molecular Physiology, LSUMC, PO Box 33932, Shreveport, LA 71130-3932.
Received 12 January 1999; accepted in final form 22 April 1999.
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