D-Serine is reabsorbed in rat renal pars recta

Stefan Silbernagl1, Katharina Völker1, and William H. Dantzler2

1 Physiologisches Institut der Universität Würzburg, D-97070 Würzburg, Germany; and 2 Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051


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ABSTRACT
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D-Serine normally contributes up to 3% to total plasma serine and up to 23% in chronic renal failure. D-Serine is metabolized by tubular D-amino acid oxidase (D-AAO), and high D-serine plasma levels are nephrotoxic; both events are localized in the straight part of the proximal tubule. We therefore investigated if and how D-serine is reabsorbed there. We microinfused 14C-labeled D- or -L-serine + [3H]inulin into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons and into long loops of Henle (LLH) of rats in vivo and in situ. The fractional reabsorption (FR) of the 14C label was determined from the 14C:3H ratio in the final urine. At 0.36 mM, FR of D-[14C]serine was 86% (EP), 90% (LP), and approx 0 (ED, LLH). FR of D-serine could be saturated and inhibited by L-serine (and vice versa). D-methionine, but not D-glutamate or D-arginine, blocked FR of D-serine (LP). We conlude that filtered D-serine is able to enter the pars recta cells, thereby getting access to D-AAO. The uptake carrier has a very low stereospecificity and is, therefore, different from that in the proximal convolution. The colocalization of exclusive reabsorption and metabolism makes the pars recta the tubule site for the recycling of the carbon structure of D-amino acids and, at the same time, the target of D-serine nephrotoxicity.

kidney; D-amino acid transport; nephrotoxicity; Henle's loop


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INTRODUCTION
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D-SERINE IS A NORMAL plasma constituent in mice (18) and humans (3, 17). It stems from spontaneous in vivo racemization of aged proteins of long-lived tissue (20), from brain cells releasing D-serine (12), and from uptake of cooked or otherwise processed dietary proteins (7, 14). A bacterial source of D-serine has been discounted as D-serine does not occur in bacterial walls and has been found in mice living under germ-free conditions (16). D-Serine contributes 0.5% to 3% to total plasma serine in normal volunteers and up to 23% in patients with highly elevated serum creatinine levels (3, 17). This increase of plasma D-serine concentrations in chronic renal failure suggests that the kidney keeps the plasma D-serine level low. Metabolism of D-serine by tubular D-amino acid oxidase (D-AAO) (18) seems to be the main mechanism by which the kidney fulfills this role.

High doses of intraperitoneally injected D-serine damage rat proximal straight tubules within several hours, causing acute tubular necrosis with proteinuria, glucosuria, and generalized hyperaminoaciduria (4, 5, 10, 15, 19, 31, 32). From these observations, two main questions arise: 1) how does D-serine enter the cells of the proximal straight tubule, and 2) why is it toxic for these cells? The first question is addressed in this paper, whereas parallel reports from our laboratory deal with the second one (24, 25).

We demonstrated earlier that, in contrast to its L-isomer, D-serine is not reabsorbed significantly if microperfused through proximal convoluted tubules in the rat kidney in vivo and in situ (27). Transport studies with membrane vesicles prepared from rabbit renal outer cortex and from outer stripe of outer medulla (13) showed that luminal cortical membrane vesicles accumulate L- and D-serine by a single common transport system, which has a much lower affinity (Km = 30 mM) for D- than for L-serine (Km = 3.7 mM). Luminal membrane vesicles from the outer medulla take up L-serine by two kinetically distinct processes (Km = 0.37 and 10 mM, respectively), whereas D-serine transport is not detected in these vesicles (13). Uptake of L-serine into basolateral vesicles has been demonstrated too. It can be inhibited by L-phenylalanine, but not by D-serine (13). Shimomura et al. (21) microperfused rabbit proximal nephron segments in vitro and found a high intracellular accumulation of the radiolabel of L-serine and, to a smaller extent, of D-serine if they were offered at the basolateral side. Accumulation was similar in the convoluted and straight portions of the proximal tubule (21). Thus, in rabbits, there is some evidence from in vitro experiments that D-serine can enter the cells of the proximal tubule not only by the same luminal carrier as the L-isomer, albeit at a lower affinity, but also via a basolateral carrier.

As D-serine nephrotoxicity is localized in the straight part of the proximal tubule (5), and as renal D-serine metabolism by D-AAO is localized there (6, 29), we wanted to investigate whether, in contrast to the proximal convolution (27), D-serine is reabsorbed in the straight part. For this purpose, we microinfused 14C-labeled D- or L-serine into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons, as well as into the long loops of Henle (LLH) of juxtamedullary nephrons of the rat in vivo and in situ and determined the fractional recovery of the 14C label compared with co-microinfused 3H-inulin in the final urine.


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Male Munich-Wistar rats were used for the following groups of experiments: 1) EP, LP, and ED experiments [shown in Figs. 2 to 5; 198-357 g body wt (269 g mean body wt); Harlan Sprague Dawley, Indianapolis, IN]; 2) LP experiments [shown in Fig. 6; 152-382 g body wt (275 g mean body wt); Charles River, Sulzfeld, Germany]; and 3) LLH experiments [120-143 g body wt (136 g mean body wt); Charles River]. Group 1 was fed on Teklad 4% Mouse/Rat Diet 7001; groups 2 and 3 were fed on Altromin Standard Diet 1320. All groups had free access to water. The animals were anesthetized with Inactin (120 mg/kg body wt; Byk-Gulden, Konstanz, Germany). A tracheostomy was performed and polyethylene cannulae were placed in the right jugular vein for infusions. The animals were infused with Ringer solution at a rate of 0.15 ml · min-1 · kg body wt-1. The Ringer solution contained the following (in g/l): 9 NaCl, 0.4 KCl, 0.25 CaCl2, and 0.2 NaHCO3. We prepared the kidney for tubule micropuncture using standard techniques (1)

Microinfusion into superficial nephrons. After identification of the nephron segments by intravenous injection of lissamine green SF (Chroma-Gesellschaft; Schmidt, Köngen, Germany) at a dose of 0.02 ml of a 100 g/l solution titrated with NaOH to pH 7.4, we micropunctured the tubule using glass capillaries. The latter had ground tips (outer tip diameter: 9-11 µm) and were mounted on a microperfusion pump (28). Microinfuson sites were 1) the first superficial loop of the proximal tubule (EP), 2) the last superficial loop of the proximal tubule (LP), and 3) the first superficial loop of the distal tubule (ED) (see Fig. 1). In all cases, the microinfusate (10 nl/min) added to the endogenous flow rate of tubular fluid. The microinfusate (pH 6.7) contained (in g/l) 9 NaCl, 0.4 KCl, 0.25 CaCl2, and 2.0 MOPS, as well as 7.4 GBq/l [3H]inulin (5.18 GBq = 140 mCi/g; American Radiolabeled Chemicals, St. Louis, MO), 0.36 mmol/l 1-14C-labeled D-serine (2.04 GBq = 55 mCi/mmol; American Radiolabeled Chemicals), or 14C(U)-labeled L-serine (6.2 GBq = 167 mCi/mmol; New England Nuclear, Boston, MA; or 2.04 GBq = 55 mCi/mmol; American Radiolabeled Chemicals), in addition to unlabeled D- or L-serine, D-methionine, D-glutamate, or D-arginine, as indicated in RESULTS. Microinfusion lasted for 10 min. Starting shortly before microinfusion, the ipsilateral urine was collected from the ureteral catheter in 15-min fractions for 1 h, and the 14C and 3H counts (in dpm) of each fraction were determined in a liquid scintillation spectrometer (LS 6000SE; Beckman, Anaheim, CA; or 1600 TR; Canberra-Packard, Frankfurt am Main, Germany). To be sure that the 14C activity found in the urine still represented serine, 14C-containing urine samples, as well as the microinfusion fluid, were analyzed by HPLC (25a), and 1-min fractions of the outflowing eluent were collected in scintillation vials. The 14C counts of each fraction were determined in a liquid scintillation spectrometer. As a result, the radiochemical purity of the microinfusion fluid was >95%. In the urine samples, it was >90%. As a further control, the urine of the contralateral kidney was collected from a bladder catheter in 15-min fractions during the same 1-h period. The [3H]inulin counts (if any) and the 14C-labeled D- or L-serine counts in the contralateral urine, never exceeding 10% of those in the ipsilateral urine, were subtracted from the latter. After this correction, the fractional recovery was calculated from the sum of the 14C and 3H counts (in dpm), respectively, of the 1-h collecting period, as shown in Fig. 1.


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Fig. 1.   Microinfusion technique. Solutions containing 14C-labeled D- or -L-serine (14Cinf) and [3H]inulin (3Hinf) were microinfused (10 nl/min for 10 min) by microperfusion pump (28) into first superficial loop of proximal tubule [early proximal (EP)], last superficial loop of proximal tubule [late proximal (LP)], first superficial loop of distal tubule [early distal (ED)], and long loops of Henle (LLH), near their hairpin bend. Starting shortly before microinfusion, ipsilateral urine was collected from ipsilateral final urine in 15-min fractions for 1 h, and 14C and 3H dpm were counted (14Curine and 3Hurine). For details, see METHODS.

Microinfusion into LLH. The experiments on LLH were performed as described previously (8, 9). Briefly, the papilla of the left kidney was exposed and a single ascending limb of the LLH was punctured near the hairpin bend with a glass micropuncture pipette with an external tip diameter of 5-6 µm and mounted to a microperfusion pump (28). The tip of this pipette was coated with platinum glaze to make it easily visible (11). The loop was then infused with a TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid)-buffered (10 mmol/l) solution containing [3H]inulin and D-[14C]serine (7.28 mmol/l), as described above. The microinfusion solution also contained lissamine green (20 g/l), so that the flow in the loop could be seen and so that we could determine whether there was any extravasation from the loop that would make the infusion technically unacceptable. The microinfusion was generally maintained at 10 nl/min. After the microinfusion was well established (usually 2-3 min), we made collections of urine emerging from the ducts of Bellini with a second micropuncture pipette (external tip diameter: 12-14 µm). The urinary volume recovery at this collection site was much smaller than that obtained from the ureteral catheter during microinfusion into superficial nephrons. Therefore, the lowest D-[14C]serine concentration used had to be higher (7.28 mmol/l) than that used for superficial nephrons (0.36 mmol/l). The radioactivity in the collected fluid and the initial perfusion solution was measured in a liquid scintillation counter (1600 TR; Canberra-Packard) to determine the fractional recovery of the infused D-[14C]serine in the urine of the ducts of Bellini. Two to five collections of approx 70-100 nl were made in each infusion experiment (the number depending on the length of time the infusion could be maintained), and the mean value for the fractional recovery for all collections was used as the value for that microinfusion experiment.

Chemicals. The D- and L-serine, D-methionine, D-glutamate, and D-arginine were purchased from Sigma (Deisenhofen, Germany), TES and MOPS were obtained from Serva (Heidelberg, Germany), and all other chemicals were from Merck (Darmstadt, Germany).

Calculations and statistics. The maximal reabsorption rate (Jmax; pmol/s per loop of Henle) and the apparent Michaelis constant (mmol/l) of D-serine reabsorption from short loops of Henle were estimated from the mean reabsorption rates (J) at the concentrations (C), whereby J = (1 - recovery) × microinfusion rate × C (pmol/s per loop of Henle), by the three linearized plots of the Michaelis-Menten equation (1/J vs. 1/C, C/J vs. C, and J vs. J/C).

All results shown in Figs. 2-6 are summarized there as means ± SE (n = number of microinfused nephron segments). The level of significance for differences between means of unpaired observations was determined with Student's t-test. Differences assessed by t-test were considered statistically significant at P < 0.05. 


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Fig. 2.   Fractional 14C recovery in final urine during microinfusion (10 nl/min) of 0.36 or 7.28 mmol/l D-[14C]serine into EP, LP, and ED segments of superficial nephrons, as well as of LLH of juxtamedullary nephrons. Values are means ± SE; n = no. of tubules (shown in parentheses).


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ABSTRACT
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High doses (7.5 mmol/kg body wt) of D-serine, leading to D-serine plasma concentrations of 12 to 27 mmol/l within 15 min in Wistar rats (unpublished obervations), are nephrotoxic and cause a generalized hyperaminoaciduria within several hours (see INTRODUCTION). As similarly high D-serine concentrations were used in the present microinfusion experiments, lasting for 10 min, the early time course of leucine (Leu) and ornithine (Orn) excretion was determined in pilot experiments. As a result, fractional excretion (FE) of these amino acids (FE control: Leu and Orn < 1%) did not significantly increase after 10 min and started to rise to severe values (>5%) only after 60 min. Thus significant toxic effects of microinfused D-serine can be excluded, to a great extent, in the present experiments.

Figures 2-6 show the fractional recovery (see METHODS) of the 14C label of D- and L-serine (i.e., the original data). As the reabsorptive process, rather than excretion, is the major focus of this paper, the following results are presented as fractional reabsorption [FR (%) = 100 - fractional recovery (%)], i.e., the distance on the ordinate between the height of the columns and 100% (Figs. 2-6).

In the first series of experiments, we microinfused a solution (10 nl/min) containing 0.36 mmol/l 14C-labeled D-serine into EP, LP, and ED tubule sections appearing at the surface of the kidney, and a solution containing 7.28 mmol/l 14C-labeled D-serine into the ascending limb of an LLH near the hairpin bend. As can be seen from Fig. 2, FR of D-[14C]serine was ~86% during EP, ~90% during LP, but essentially zero during ED, as well as during LLH microinfusion. The ED and LLH values for fractional recovery were not significantly different from 100%. Thus D-serine is nearly completely reabsorbed if it is microinfused upstream from the short loops of Henle at an initial concentration of 0.36 mmol/l, but it is not reabsorbed at all downstream of the hairpin bend of the LLH (7.28 mmol/l). Therefore, the tubule section beyond the last accessible loop of the proximal tubule (i.e., the pars recta) is responsible for the high D-serine FR in the loop of Henle.

To evaluate, in further sets of experiments, whether the high reabsorption observed during EP and LP microinfusion was carrier mediated, we repeated the experiments with the highly elevated D-serine concentrations of 20 and 80 mmol/l. As shown in Fig. 3, FR of D-serine during EP microinfusion decreased from 86% at 0.36 mmol/l to 70% (n = 3) at 20 mmol/l and to 22% (n = 5) at 80 mmol/l. Similar to these EP values, FR of D-serine during LP microinfusion decreased from 90% at 0.36 mmol/l to 55% (n = 6) at 20 mmol/l and to 21% (n = 6) at 80 mmol/l. These data demonstrate that at least the major part of D-serine reabsorption from the pars recta reflects mediated transport.


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Fig. 3.   Fractional 14C recovery in final urine during microinfusion (10 nl/min) of 0.36 mmol/l D-[14C]serine [in presence or absence of 80 mmol/l L-serine (L-Ser)], as well as of 20 or 80 mmol/l D-[14C]serine into EP and LP segments of superficial nephrons. Values are means ± SE; n = no. of tubules (shown in parentheses). ** P < 0.01.

Apparent kinetic parameters roughly estimated from the LP values at 0.36, 20, and 80 mmol/l (Fig. 3) were as follows: Jmax = 3.80 ± 0.24 pmol/s per pars recta (SD); concentration at 1/2 Jmax (Km) = 24.1 ± 2.5 mmol/l (SD); and Hill coefficient = 0.99 (see DISCUSSION).

To test whether L-serine had any effect on D-serine reabsorption, we repeated the EP and LP microinfusion experiments with 0.36 mmol/l D-[14C]serine in the presence of 80 mmol/l L-serine in the microinfusate (Fig. 3). During EP microinfusion, the FR of 0.36 mmol/l D-[14C]serine in the presence of 80 mmol/l L-serine (31%) was not significantly different from that in the presence of 80 mmol/l D-serine (22%). During LP microinfusion, however, the FR of D-[14C]serine (0.36 mmol/l) was completetely inhibited by 80 mmol/l L-serine. These results demonstrate that 1) L-serine inhibits D-serine reabsorption in the pars recta, and 2) D-serine reabsorption is an entirely mediated process.

In a further set of experiments (Fig. 4), we examined the FR of L-[14C]serine (0.36, 20, and 80 mmol/l) during EP and LP microinfusion. During EP, as well as during LP microinfusion (compare Figs. 3 and 4), L-serine was reabsorbed to about the same high extent (92% for EP; 89% for LP) as the D-isomer (86% for EP; 90% for LP) at 0.36 mmol/l. At 80 mmol/l, however, the results with L-serine were quite different from those with D-serine. During EP microinfusion, ~55% of L-[14C]serine was reabsorbed (Fig. 4), compared with only ~22% of D-[14C]serine (Fig. 3). During LP microinfusion at 80 mmol/l, however, FR of L-[14C]serine dropped to only 4% (Fig. 4), compared with 21% in the case of D-[14C]serine (Fig. 3).


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Fig. 4.   Fractional 14C recovery in final urine during microinfusion (10 nl/min) of 0.36 mmol/l L-[14C]serine [in presence or absence of 80 mmol/l D-serine (D-Ser)], as well as of 20 or 80 mmol/l L-[14C]serine into EP and LP segments of superficial nephrons. Values are means ± SE; n = no. of tubules (shown in parentheses).

We also tested whether D-serine (80 mmol/l) had any effect on the reabsorption of the L-isomer (0.36 mmol/l). During EP microinfusion, L-[14C]serine FR was only slightly diminished by D-serine (83% vs. 92% in the control), whereas during LP microinfusion, FR of L-[14C]serine dropped from 89% (control) to 35% (Fig. 4). These data confirm our earlier conclusion that the serine-transporting carriers of the proximal convoluted tubule (27) highly prefer the L-isomer. This results in a higher capacity of the whole proximal tubule to reabsorb L-serine than to reabsorb D-serine. The present data show, in addition, that D- and L-serine inhibit each other during reabsorption in the pars recta.

In a further set of LP microinfusion experiments, we evaluated the specificity of the reabsorptive mechanism for D- and L-serine in the pars recta by adding D-methionine to the LP microinfusate containing 0.36 mmol/l 14C-labeled D- or -L-serine. In the presence of 80 mmol/l D-methionine, the FR of D-[ 14C]serine decreased from 90% to 2%, but that of L-[14C]serine only decreased from 89% to 74% (Fig. 5). During EP microinfusion, there was no influence of 80 mmol/l D-methionine on the reabsorption of 0.36 mmol/l L-[14C]serine (90 ± 1% vs. 92 ± 2%, n = 4 and 5, respectively).


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Fig. 5.   Fractional 14C recovery in final urine during microinfusion (10 nl/min) of 0.36 mmol/l 14C-labeled D- or -L-serine into tubule fluid of LP segments of superficial nephrons in presence or absence of 80 mmol/l D-methionine. Values are means ± SE; n = no. of tubules (shown in parentheses).

In a final set of LP microinfusion experiments, we wanted to test the extent to which D-glutamate- or D-arginine+ inhibits the reabsorption of D-[14C]serine (0.36 mmol/l) in the pars recta. As these experiments had to be performed in Würzburg (instead of in Tucson, as in those experiments detailed above), we purchased the Munich-Wistar rats from a German dealer (see METHODS). It turned out that the FR of D-[14C]serine in the control experiments (LP; 0.36 mmol/l) was much smaller (55%; see Fig. 6) than those found with Munich-Wistar rats purchased in Tucson (90%; see Fig. 2). The FR of L-[14C]serine (LP; 0.36 mmol/l) differed as well, albeit to a smaller extent: 73% vs. 89% (see Figs. 4 and 6). We then repeated the LP microinfusion experiments with 20 and 80 mmol/l D-serine (Fig. 6). The results were again significantly different from those reported above (compare LP values in Figs. 3 and 6). Roughly estimating the kinetics in this case (see DISCUSSION), we obtained the following values: Jmax = 1.88 ± 0.38 pmol/s per pars recta (SD); Km = 17.3 ± 6.8 mmol/l (SD); Hill coefficient = 0.98. Thus Jmax of the pars recta of the Würzburg rats was significantly smaller than that of the Tucson rats, whereas the affinity constant was in the same range in both groups.


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Fig. 6.   Fractional 14C recovery in final urine during microinfusion (10 nl/min) of 0.36 mmol/l L-serine and of 0.36 mmol/l D-[14C]serine [in presence or absence of 80 mmol/l D-glutamate (D-Glu) or D-arginine (D-Arg)], as well as of 20 or 80 mmol/l D-[14C]serine into LP segments of superficial nephrons. Values are means ± SE; n = no. of tubules of Würzburg rats (shown in parentheses; see RESULTS). * P < 0.05 compared with 100% fractional recovery; ** P < 0.01; *** P < 0.001; ns, not significant.

In contrast to the results with D-methionine (Fig. 5), the FR of 0.36 mmol/l D-[14C]serine was not significantly inhibited when 80 mmol/l D-glutamate or D-arginine were added to the LP microinfusate (Fig. 6).


    DISCUSSION
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The present data demonstrate that radiolabeled D-serine is reabsorbed by a high-capacity/low-affinity process (Km approx  20 mmol/l) in the pars recta at similar rates as L-serine. This is in contrast to our recent data, obtained from rat proximal convoluted tubules microperfused in vivo and in situ, in which only the L-isomer was reabsorbed to a significant extent (27). These earlier findings were confirmed by the present results that D-[14C]serine reabsorption is inhibited by L-serine to a greater extent during LP than during EP microinjections (Fig. 3), and vice versa (Fig. 4). Both results can be explained by a much higher stereospecificity of serine reabsorption in the convoluted than in the straight part of the proximal tubule. This is somewhat at variance with the results of uptake studies with luminal membrane vesicles from outer cortical and outer medullary (outer stripe) rabbit tubules (13). These authors concluded from their results that outer cortical vesicles, stemming mostly from the pars convoluta, take up L-serine (Km = 3.7 mmol/l) and D-serine (Km = 30 mmol/l) by the same transport system, whereas the outer medullary vesicles (pars recta) do not take up D-serine at all. However, these data are hard to compare with the present findings, not only because of possible species differences, but also because 1) kinetic data estimated from vesicular uptake and from in vivo reabsorption usually differ by one or more orders of magnitude, for unknown reasons (22); 2) the outer stripe of the outer medulla does not contain only the pars recta of the proximal tubule; 3) preparations of luminal membranes usually also contain basolateral membranes, to some extent; and 4) D-AAO is absent in vesicles, but works in series with the luminal uptake in vivo. The apparent kinetic parameters roughly estimated in this study may, therefore, represent overall kinetics, including the D-serine breakdown in the cells of the pars recta (see below).

The present results, especially the observation that D-serine reabsorption is fully inhibited by high concentrations of L-serine (Fig. 3), demonstrate that D-serine reabsorption from the loop of Henle is an entirely mediated process. Whether this represents a novel carrier, or D- and L-serine are accepted by one of the known amino acid carriers to a similar extent, is not clear. Recently, Utsunomiya-Tate et al. (30) have cloned a system ASC-like, Na+-dependent neutral amino acid transporter, ASCT2, from mouse testis cDNA. Expressed in oocytes, it has a high affinity (Km approx  0.020 mmol/l) for L-alanine, L-threonine, L-cysteine, and L-glutamine. L-Alanine transport by ASCT2 can be inhibited by D-serine. Although expression of ASCT2 mRNA has been demonstrated in mouse kidney (30), it does not seem to be very likely that ASCT2 is involved in L- and D-serine reabsorption in the loop of Henle because ASTC2 has a 100-fold lower Km for L-serine than that found in this study for D-serine. Moreover, L-alanine transport by ASCT2 is only slightly inhibited by D-alanine (30), whereas D-alanine is reabsorbed in microperfused loops of Henle at the same high rate (23) as D-serine was in the present study, an observation supporting the possibility that D-alanine and D-serine share a common carrier in the pars recta, with similar transport rates.

We have recently reported that transport of taurine differs in short loops of Henle of Munich-Wistar rats coming from different sources (26). The present data, obtained with rats purchased from the same two dealers, show that this is also true for D-serine reabsorption. In both cases, the rats bought in Tucson reabsorbed the respective amino acid from short loops of Henle at a significantly higher rate than the rats purchased in Würzburg (see METHODS for sources). This time, we roughly estimated the overall kinetic constants of D-[14C]serine reabsorption and ended up with a significantly lower maximal transport rate (fmol/s per loop of Henle) in the Würzburg rats than in the Tucson rats, whereas the affinity constant was in the same range. As the microinfusate was diluted when entering the tubule lumen, the intratubular D-serine concentrations were lower than those in the microinfusion pipette and, therefore, these kinetic parameters are not very useful as absolute values. However, the difference in Jmax at unchanged Km values suggests that the density or activity of the carrier per membrane area is higher or that the tubule section involved in reabsorption is longer in Tucson rats than in Würzburg rats.

As mentioned above, reabsorption of D-amino acids in the pars recta delivers these substrates to D-AAO localized in the peroxysomes of the same nephron section (6, 29). This FAD-coupled enzyme converts neutral alpha -D-amino acids into their respective alpha -keto acids, which in turn can be used to form their respective, more useful, alpha -L-amino acids by transamination. We therefore tested whether amino acids that are accepted well (D-methionine), slightly (dibasic D-amino acids), or not at all (D-glutamate) by the renal D-AAO (2) affect D-serine reabsorption. Indeed, D-methionine is a strong inhibitor of D-serine reabsorption (Fig. 5), whereas D-arginine only has a small effect, and D-glutamate has no effect at all (Fig. 6). This could mean that 1) the carrier has a specificity similar to that of D-AAO and/or 2) D-methionine inhibits D-AAO and, thereby, competitively inhibits the metabolic sink for D-serine. In the latter case, the chemical gradient for D-serine across the luminal membrane and, therefore, its removal from the lumen would decrease. The second process seems to play a partial role, at least, because 1) reabsorption of D-alanine (another good substrate of D-AAO) in the loops of Henle is partly inhibited in the presence of the two D-AAO inhibitors, benzoate and indole carboxylic acid (23), and 2) reabsorption of LP-microinfused L-serine was much less inhibited by D-methionine than reabsorption of D-serine (Fig. 5). Although, to our knowledge, no information is available on D-serine transport across the peroxisomal membrane, competition for a carrier could take place there too.

The colocalization of D-serine metabolism and the exclusive D-serine reabsorption in the pars recta can explain why nephrotoxicity of high D-serine doses is confined to the S3 segment (4). Our recent data (24, 25) suggest that the nephrotoxicity is caused by H2O2 (and/or oxygen radicals) formed during D-serine metabolism by peroxisomal D-AAO. Because D-serine filtered at the glomerulus is not reabsorbed in the proximal convoluted tubule and is, therefore, concentrated about three times until the tubular fluid reaches the pars recta, even moderately elevated D-serine plasma levels, as observed in chronic renal failure (see INTRODUCTION), might affect the tubule cells of the pars recta.

In conclusion, D-serine is able to enter tubule cells of the pars recta from the lumen, thereby getting access to D-AAO, the enzyme responsible for the metabolism of neutral D-amino acids. D-Serine reabsorption is mediated by a carrier that has a very low stereospecificity, if any. For this reason, the carrier in the straight part is different from that or those in the proximal convolution, and might be a novel one. D-serine, once reabsorbed, ends up in the metabolic sink of D-AAO, thereby keeping up the chemical D-serine gradient directed from lumen into the cell. The colocalization of exclusive reabsorption and metabolism makes the pars recta the tubule site for recycling the carbon structure of D-amino acids and, at the same time, the target of D-serine nephrotoxicity.


    ACKNOWLEDGEMENTS

We thank Olga Brokl and Kristen Evans for encouragement and support at the University of Arizona, and Dr. Michael Gekle for valuable discussions.


    FOOTNOTES

This study was supported, in part, by North Atlantic Treaty Organization Collaborative Grant 0104/88 and National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-16294.

Parts of this study have been presented, in abstract form, at the 29th Annual Meeting of the American Society of Nephrology, New Orleans, November 1996 (J. Am. Soc. Nephrol. 7: 1305, 1996) and at the 76th Congress of the Deutsche Physiologische Gesellschaft in Rostock, March 1997 (Pflügers Arch. 433: R165, 1997).

Address for reprint requests and other correspondence: S. Silbernagl, Physiologisches Institut der Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany.

Received 1 December 1997; accepted in final form 25 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Physiol 276(6):F857-F863
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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