EPILOGUE
Glutamate transport asymmetry and metabolism in the functioning kidney

Scott Schuldt, Patsy Carter, and Tomas Welbourne

Department of Cellular and Molecular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 X-AG 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 X-AG subtypes; excitatory amino acid transporter carrier 1; GLT1 glutamate transporter; 5-oxoproline; glutamine; ammonium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 X-AG 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 X-AG 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 X-AG, 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.


    MATERIALS AND METHODS
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INTRODUCTION
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 · min-1 · 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 [alpha ]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 div  15 min) equals luminal net uptake
luminal net uptake = {GFR × [Glu]P} − {V × [Glu]U}
The fractional excretion (FE) of glutamate was calculated as excreted glutamate divided by the amount filtered
FE = V × [Glu]U ÷ {GFR × [Glu]P}
Removal rate per minute infusion period was calculated from the change in perfusate glutamate content over that period (corrected for the urinary loss) minus the infusion rate, 600 nmol/min
Removal rate = 600 nmol/min − {perfusate volume × &Dgr;[Glu]P ÷ 15 min} − {urine volume × [Glu]U ÷ 15 min}
Perfusate volume was corrected for the urine volume loss and infusate volume gain over the 15-min period. For example, if perfusate glutamate content remains unchanged during the infusion period, and if all filtered glutamate is reabsorbed, the removal rate would equal the rate of infusion. If perfusate glutamate content increases, the removal rate would be the difference between this increase minus the infusion rate. If the perfusate glutamate content decreases, this decrease added to the infusion rate equals the removal rate. The antiluminal net uptake was taken as the difference between the removal rate and luminal uptake
antiluminal net uptake = removal rate − luminal net uptake
Formation of glutamine, ammonium, and 5-oxoproline was taken as the rate of accumulation in the perfusate plus that excreted over the 45-min infusion period. Results are expressed in nanomoles per minute per kidney with an average wet weight of 1.4 ± 0.2 g per kidney. Comparisons were made between L-glutamate and D-glutamate infused alone, as well as between each infused alone vs. in combination with D-aspartate. Statistical differences were obtained using the Student's t-test. Statistical significance was determined using either a one-tailed or two-tailed t-table, depending on whether a priori directional changes were postulated on the basis of the in vivo model (4).


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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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Fig. 1.   Perfusate glutamate concentration over the 60-min perfusion time course, showing disappearance of glutamate from perfusate. Initial glutamate concentration, time 0, was ~250 µM. At 15 min, infusion of either D- or L-glutamate (Glu) alone or in combination with D-aspartate (Asp) at the rate of 600 nmol/min was started and maintained for 45 min. Points are means ± SE from 4-6 kidneys/group; error bars for D-aspartate-coinfused experiments not shown (SE values for D-glutamate are 4, 24, 44, 48, and 34, and for L-glutamate 10, 18, 30, 60, and 22 at 0, 15, 30, 45, and 60 min, respectively). * Differences between glutamate isomers; ** differences between glutamate- and D-aspartate-infused kidneys, P < 0.05.

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 X-AG on the luminal surface (25, 26), and specifically subtype EAAC1 (25).

                              
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Table 1.   Removal rates for L- and D-glutamate



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Fig. 2.   Luminal uptake rates for L- and D-glutamate alone or with D-aspartate (D-A) over the 45- to 60-min infusion period. Uptake rates are the difference between filtered and excreted glutamate (see MATERIALS AND METHODS for calculations). Results are means ± SE from 4-6 kidneys/group. * Differences between glutamate isomers; ** differences between glutamate and D-aspartate-infused kidneys, P < 0.02.



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Fig. 3.   Antiluminal glutamate uptake rates for L- and D-glutamate. Antiluminal uptake rate was taken as the difference between removal rate and luminal uptake rate. Measurements are the average of 3 consecutive infusion periods, with results shown as means ± SE; * Differences between glutamate isomers; ** differences between glutamate and D-aspartate-infused kidneys, P < 0.02.

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 X-AG 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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Table 2.   Glutamate uptake and release of metabolites

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 X-AG 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 X-AG 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 X-AG 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 gamma -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.


    ACKNOWLEDGEMENTS

We thank Tian Ma and Josh Chance for excellent technical support, and we acknowledge Genentech for funding.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(3):E439-E446
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