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Am J Physiol Renal Physiol 293: F382-F390, 2007. First published April 11, 2007; doi:10.1152/ajprenal.00441.2006
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Why is D-serine nephrotoxic and {alpha}-aminoisobutyric acid protective?

Alexander W. Krug,1 Katharina Völker,1 William H. Dantzler,2 and Stefan Silbernagl1

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

Submitted 6 November 2006 ; accepted in final form 4 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
D-Serine selectively causes necrosis of S3 segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid {alpha}-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S3, thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of D-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to ~30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[14C]serine or [14C]AIB (0.36 mmol/l) together with [3H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[14C]serine and [14C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated D-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents D-serine-induced tubulopathy by inhibition of D-serine uptake in S3 segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.

rat; kidney; D-serine toxicity; glutathione

THE NORMAL D-SERINE plasma concentration in healthy human individuals is 1.4–1.6 µmol/l, which is a 0.015 fraction of the L-isomer concentration (D/L ratio; see Refs. 5, 17, and 31). Exogenous D-serine from cooked or industrially processed food and antibiotics as well as from in vivo racemization of aged proteins contributes to the D-serine plasma level. In addition to these exogenous sources, relatively high amounts of endogenously generated D-serine were found in human brains (11, 24, 34) and to some extent also in the final urine (18).

D-Serine is considered to be an agonist at the glycine-binding site of N-methyl-D-aspartate (NMDA) receptors (13, 24, 35). Therefore, because NMDA receptor malfunction is postulated to play a role in the pathophysiology of schizophrenia, D-serine (30 mg = 0.28 mmol/kg body wt) has been added to established antipsychotic drugs in the treatment of schizophrenic patients (9, 16, 18, 35, 42, 43,). In these patients, D-serine plasma levels were elevated up to 100–150 µmol/l, 100 times the physiological concentrations (16, 43).

Concern about using D-serine therapeutically, however, arises because high doses of intraperitoneally injected D-serine damage rat proximal straight tubules (S3 segment) within several hours of administration, causing acute tubular necrosis with proteinuria, glucosuria, and generalized hyperaminoaciduria (2, 6, 7, 14, 30, 46). This tubulotoxic effect can be prevented by coinjection of certain amino acids (4, 21, 31), e.g., {alpha}-aminoisobutyric acid (AIB; see Refs. 21 and 23). Thus questions arise as to why D-serine is toxic for these cells and how AIB is able to protect them.

In contrast to its L-isomer, D-serine is not reabsorbed significantly if microperfused through the proximal convoluted tubule of the rat kidney in vivo et situ (39). Because D-serine nephrotoxicity is localized in the straight part of the proximal tubule (6, 7) and because renal D-serine metabolism by D-amino acid oxidase (D-AAO) is localized there also (8, 22, 25, 44), we have recently investigated (38, 41) whether, in contrast to the proximal convolution (39), D-serine is reabsorbed by a specific carrier in the straight part. We concluded from our results that filtered D-serine is able to enter the pars recta cells across their apical membrane (38, 41). The apical uptake carrier has a very low stereospecificity for serine (41) and alanine (10, 38) and is, therefore, different from that in the proximal convolution (39). The colocalization of exclusive reabsorption and metabolism of D-serine 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.

D-AAO is the only enzyme known to metabolize D-amino acids in the kidney (29) and seems to be the main mechanism by which the kidney eliminates D-serine (32). D-AAO metabolizes D-amino acids into their corresponding {alpha}-keto acids and NH3 (Fig. 1 and Refs. 15 and 25). Regeneration of the coenzyme flavine-adenine-dinucleotide from the reduced to the oxidized state generates cytotoxic H2O2, which in turn can produce even more aggressive reactive oxygen species (ROS) such as HO2· or O2bullet– (Fig. 1). Physiological detoxification enzymes like glutathione peroxidase or catalase protect the cells from radical-induced damage.


Figure 1
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  		Fig. 1. Hydrogen peroxide formation from D-amino acids and oxygen radicals and disposition by catalase and glutathione (GSH) peroxidase.

 
We hypothesize that excessive D-serine-induced H2O2 generation (Fig. 1) might interfere with physiological detoxification processes, leading to a decrease in renal cellular content of reduced glutathione (GSH), to subsequent massive generation of ROS, and consequently to oxidative destruction of cells in the S3 segment. If this is true, D-serine-induced hyperaminoaciduria and glucosuria should be accompanied by a lowered GSH concentration in renal tissue. Furthermore, an exogenous GSH supply should reduce D-serine-induced hyperaminoaciduria and glucosuria.

AIB is a nonmetabolizable, neutral amino acid that is structurally related to D-serine. AIB, if coinjected with D-serine in equimolar doses, protects the S3 segment against D-serine-induced tubule necrosis (21, 23). This protective effect might be caused by 1) interference of AIB with the D-AAO-mediated oxidation of D-serine within the tubule cell and/or 2) an inhibitory influence of AIB on apical D-serine uptake in the cells of the S3 segment.

To test these hypotheses, the following series of experiments were performed. 1) The time course and the dose-response relationship of D-serine-induced hyperaminoaciduria and (partly) of glucosuria in rats were evaluated. 2) GSH concentrations in renal tissue before and after intraperitoneal injections of D-serine were measured. 3) The effect of coinjection of GSH on D-serine-induced hyperaminoaciduria and on renal GSH concentrations was determined. 4) Reabsorption of [14C]AIB or D-[14C]serine in short loops of Henle in the absence and presence of high concentrations of D-serine and AIB was evaluated by tubular microinfusion in vivo et situ. 5) The specific activity of D-AAO with different concentrations of D-serine as substrate and in the presence and absence of 80 mmol/l AIB was determined in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male Munich Wistar rats (body wt 210–350 g; Charles River, Sulzfeld, Germany) had free access to water and were fed with Altromin Standard Diet No. 1320. The protocol of the animal research performed in this study was submitted and approved by the Animal Protection Commission of the Government of Lower Franconia/State of Bavaria. The animals were anesthetized with Inactin (120 mg/kg body wt; Byk-Gulden, Konstanz, Germany) and infused with Ringer solution at a rate of 0.15 ml·min–1·kg body wt–1. The Ringer contained the following (in g/l) 9 NaCl, 0.4 KCl, 0.25 CaCl2, and 0.2 NaHCO3. A tracheostomy was performed, and polyethylene cannulas were placed in the right jugular vein for infusions, in the bladder for urine collections, and in the right femoral artery for blood collections.

Clearance studies. For the clearance experiments, 2 g/l FITC-labeled inulin (Bioflor, Uppsala, Sweden) were added to the Ringer solution (see Ref. 40 for details). Urine was collected using calibrated glass capillaries, and the urinary flow rate was estimated by taking the filling time. Blood was taken in the middle of each urine-collecting period.

Urine and blood samples were taken before (time 0, control) and after (2 and 4 h) the injection of different D-serine doses administered intraperitoneally at concentrations of 0.25, 0.76, 2.54, or 7.6 mmol/kg body wt. To investigate the effect of AIB on D-serine nephrotoxicity (at 7.6 mmol/kg body wt D-serine), an equimolar AIB dose was coinjected (at time 0). To maintain stable AIB plasma levels throughout the whole experiment, 1.3 g AIB/100 ml Ringer was added to the infusion solution after taking control blood and urine samples.

Micropuncture studies. The kidney was prepared for tubule micropuncture using standard techniques (1). Our microinfusion technique has been described recently (10, 40). We microinfused D-[14C]serine or [14C]AIB (0.36 mmol/l) together with [3H]inulin in the absence or presence of 80 mmol/l AIB or D-serine in late proximal nephrons and collected the final urine to measure the fractional recovery (FR) of the 14C activity therein. The microinfusion (10 nl/min) in the late proximal tubule lasted for 10 min. Ipsilateral (and for control, contralateral) urine for counting disintegrations per minute was collected for 1 h after the onset of microinfusion.

Detection of glucose, amino acids, inulin and GSH. Glucose was detected as NADH after enzymatic conversion to 6-phosphogluconate (Infinity Glucose Reagent no. 17500P; Sigma, Deisenhofen, Germany). Amino acids in plasma and urine were determined by HPLC (40). FITC-labeled inulin was measured fluorometrically. GSH in renal tissue was determined colorimetrically (GSH-400 method; Bioxytech, Bonneuil, France).

D-AAO activity. D-AAO activity with 8 mmol/l D-serine as a substrate was measured photometrically (3, 4, 31) in the presence and absence of 80 mmol/l AIB. In brief, D-serine was converted by D-AAO into hydroxypyruvate and H2O2. The latter was peroxidase-oxidized, thereby converting the chromophore {sigma}-dianisidine into its reduced form, which was detected at 435 nm. D-AAO (2 IU/200 µl) reaction mixture was used in PBS (50 mmol/l) at 37°C, pH = 8.5. After addition of AIB, the reaction medium was pH adjusted (pH = 8.5). A possible effect of AIB on peroxidase activity was excluded by testing peroxidase activity alone with 10 nmol/l H2O2 as substrate in the presence and absence of 80 mmol/l AIB.

Radiochemicals and chemicals. D-[14C]serine (2.04 GBq/mmol), [14C]AIB (2.07 GBq/mmol), and [3H]inulin (7.4 GBq/mmol) were obtained from American Radiolabeled Chemicals. D-Serine, AIB, and D-AAO were purchased from Sigma (Deisenhofen, Germany), and 3-(N-morpholino)propanesulfonic acid (MOPS) was from Serva (Heidelberg, Germany). All other chemicals came from Merck (Darmstadt, Germany).

Calculations and statistics. D-AAO kinetics were determined by the "least square fit method" using Sigma Plot 4.0 (Jandel Scientific). All results shown are summarized as means ± SE (n, no. of experiments). Significance of difference was determined with Student's t-test or ANOVA followed by the post hoc Scheffé's test (using PROPHET 5.0) as applicable; statistical significance was approved at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Time course and dose-response relationship. The D-serine dose used in earlier studies on D-serine toxicity in rats has generally been 7.6 mmol/kg body wt ip (6, 7, 36). Our present experiments performed with this dose showed that the serine plasma concentration rose above 12 mmol/l within about 30 min and fell below 2 mmol/l within the subsequent 90 min (Fig. 2). Fractional excretion (FE) of amino acids other than serine started to increase within 10 min after D-serine injection (see Fig. 3 for valine and methionine), but continued to rise for at least 8 h (Fig. 4). Thus, within the first 2 h, toxic effects of D-serine on the tubular epithelium could not be easily distinguished from direct inhibition of amino acid reabsorption by D-serine.


Figure 2
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  		Fig. 2. Serine plasma concentration before and up to 8 h after ip injection of 7.6 mmol/kg body wt D-serine (single experiment).

 

Figure 3
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  		Fig. 3. Fractional excretion of valine and methionine (±SE; n = 4) before and up to 2 h after ip injection of 7.6 mmol/kg body wt D-serine.

 

Figure 4
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  		Fig. 4. Fractional excretion of glutamine before and up to 8 h after ip injection of 7.6 mmol/kg body wt D- or L-serine (two single experiments).

 
To study the tubular toxicity of D-serine, we therefore determined FE of amino acids and glucose before (control) and 2 and 4 h after intraperitoneal D-serine injection (Tables 1 and 2, respectively). To determine also the dose dependency of D-serine toxicity, we injected four different D-serine doses, i.e., 0.25, 0.76, 2.54, and 7.6 mmol/kg body wt, and measured FE of 11 different amino acids and glucose as parameters of D-serine toxicity. As shown in Table 1, 2 h after injection of 0.25 and 0.76 mmol/kg body wt D-serine, neither the amino acids (except serine) nor glucose was significantly hyperexcreted. After administration of 2.54 and 7.6 mmol/kg body wt of D-serine, however, all determined amino acids were moderately hyperexcreted. However, amino acid excretion was not significantly changed by increasing the D-serine dose from 2.54 to 7.6 mmol/kg (P > 0.05). Table 1 also shows that glucose excretion remained unchanged 2 h after injection of 0.25 and 0.76 mmol/kg body wt D-serine but was moderately increased after injection of 2.54 and 7.6 mmol/kg body wt.


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  		Table 1. Fractional excretion of amino acids 2 h after injection of D-Serine

 

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  		Table 2. Fractional excretion of amino acids 4 h after injection of D-serine

 
Four hours after injection of the smallest dose (0.25 mmol/kg body wt D-serine), amino acid excretion was unchanged compared with control (Table 2), whereas 0.76 mmol/kg D-serine induced an elevated FE of glutamate, asparagine, serine, lysine, and glycine. At a dose of 2.54 mmol/kg body wt, D-serine caused a massive, generalized hyperaminoaciduria after 4 h. None of the measured amino acids showed a further significant increase in their FE 4 h after injection of 7.6 mmol/kg body wt D-serine compared with the 2.54 mmol/kg values. Table 2 shows that a mild glucosuria was present 4 h after injection of 0.76 mmol/kg body wt D-serine. Higher doses led to massive glucosuria 4 h after the injection.

Taking all data together, we can summarize that injection of high doses of D-serine (2.54 and 7.6 mmol/kg body wt) induced massive, generalized aminoaciduria. No pattern of specific amino acids hyperexcreted was observed either 2 or 4 h after injection of these doses of D-serine.

Effect of D-serine on renal GSH concentrations. Because GSH is consumed during D-serine metabolism by D-AAO (see Fig. 1), we measured the concentration of GSH in cortical and medullary tissue of rat kidney 4 h after intraperitoneal injection of D-serine (7.5 mmol/kg body wt). Figure 5 shows that the cortical and medullary GSH concentrations in the D-serine-treated animals were 46 and 50%, respectively, of the cortical and medullary concentrations in the control animals. If GSH was infused alone for 30 min (5 µmol/min) and then coinjected (3.3 mmol/kg body wt) with D-serine (7.5 mmol/kg body wt), the GSH concentrations in renal tissue stayed the same as under control conditions (Fig. 5). Thus D-serine treatment halved the renal GSH content, and GSH injected before and together with D-serine prevented this decrease. In the absence of D-serine injection, GSH administration did not change the renal GSH concentrations significantly (Fig. 5).


Figure 5
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  		Fig. 5. Renal cortical and medullary concentrations (mmol/kg wet wt) of GSH before (control) and 4 h after ip injection of 7.5 mmol/kg body wt D-serine (in presence or absence of coinjected equimolar GSH) or after injection of GSH alone. Nos. in brackets = no. of experiments. Significant differences (P < 0.05, ANOVA with Scheffé's test) were found for column 1 vs. columns 2–4 and 6; for column 7 vs. columns 3 and 4; for column 5 vs. columns 3 and 4; and for column 4 vs. columns 2, 6, and 8.

 
Effect of GSH on D-serine-induced hyperaminoaciduria. If the decreased GSH concentration in the kidney after D-serine injection causes directly or indirectly the tubular damage in D-serine nephrotoxicity, coinjection of equimolar amounts of GSH might reduce the hyperaminoaciduria. To test this, we determined the FE of 18 amino acids before (control) and 2 or 4 h after injection of D-serine, of D-serine + GSH, and of GSH alone. As expected (6, 7), injection of D-serine alone caused a tremendous hyperexcretion of all 12 amino acids determined (shown for valine and methionine in Fig. 6, A and B). Table 3 shows the results for 10 more amino acids 4 h after injection. FE of all amino acids rose tremendously. GSH treatment prevented this hyperaminoaciduria to a great extent. Except for taurine and glutamine, FE stayed below 2.2%. Thus D-serine-induced hyperaminoaciduria could be prevented to a great extent if GSH was given before and during D-serine treatment.


Figure 6
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  		Fig. 6. Fractional excretion of valine (A) and methionine (B) before (control) and 2 and 4 h after ip injection of 7.6 mmol/kg body wt D-serine in presence or absence of coinjected GSH. (See Table 3 for more amino acids.) Nos. in brackets = no. of experiments.

 

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  		Table 3. Fractional excretion of amino acids before (control) or 4 h after ip injection of 7.6 mmol/kg body wt D-serine in presence or absence of GSH

 
Effect of coinjected AIB on D-serine-induced hyperaminoaciduria. AIB is a nonmetabolized, neutral amino acid that is structurally related to D-serine. AIB, if coinjected with D-serine in equimolar doses, protects the S3 segment against D-serine-induced tubule necrosis (21). In a first step, we wanted to reproduce the tubuloprotective AIB effect by monitoring FE of amino acids and glucose. For this purpose, FEs of amino acids and glucose were measured 4 h after injection of 7.6 mmol/kg D-serine alone, of both D-serine and AIB together, or of AIB alone. The results are shown in Table 4: AIB prevented the massive, generalized D-serine-induced aminoaciduria and glucosuria when AIB was coinjected with D-serine. FEs of arginine, citrulline, glutamate, glutamine, isoleucine, leucine, methionine, ornithine, phenylalanine, and glucose were at control levels after coinjection. Alanine, asparagine, aspartate, glycine, lysine, and serine were slightly hyperexcreted under these conditions. However, the hyperexcretion of these amino acids after coinjection was much lower than that after injection of D-serine alone. Injection of AIB alone increased excretion of the same group of amino acids. The extent of mild hyperexcretion of these amino acids was not different after injection of AIB or of AIB + D-serine (P > 0.05). Thus we conclude that the mild hyperexcretion of alanine, asparagine, aspartate, glycine, lysine, and serine was induced by AIB and did not represent a D-serine-induced toxic effect.


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  		Table 4. Fractional excretion of amino acids/glucose 4 h after injection of D-serine, D-serine and AIB, or AIB alone

 
From these findings, the question arises how AIB protects the S3 segment against the D-serine-induced tubulopathy. Does it 1) influence intracellular D-AAO-mediated, free radical-generating D-serine metabolism and/or 2) interfere with D-serine reabsorption in the S3 segment?

D-AAO activity in vitro in absence and presence of AIB. The peroxisomal D-AAO converts D-serine into its corresponding {alpha}-keto acid, thereby generating excessive amounts of ROS that can lead to tubular destruction (Fig. 1). However, in a further series of experiments, we tested whether the protective effect of AIB on D-serine nephrotoxicity can be at least partly explained by direct interference of AIB with intracellular D-serine oxidation. In a first step, we characterized the kinetic parameters of D-AAO-activity in vitro with increasing concentrations of D-serine as substrate. From the data of Fig. 7, we estimated a maximal velocity of 2.0 µmol/mg protein and a Michaelis constant (Km) of 11 mmol/l, indicating a low substrate affinity.


Figure 7
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  		Fig. 7. Specific activity of D-amino acid oxidase (D-AAO) in vitro with different concentrations of D-serine as substrate.

 
To test whether AIB can influence the D-serine nephrotoxicity by inhibition of D-AAO directly, we determined D-AAO activity (8 mmol/l D-serine) before and after addition of 80 mmol/l AIB to the incubation media. Figure 8 shows that AIB has no significant effect on D-AAO-activity in vitro. Thus it seems very unlikely that direct interference of AIB with the enzyme activity in vivo is the reason for the protective effect of AIB on D-serine nephrotoxicity.


Figure 8
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  		Fig. 8. D-AAO activity in vitro determined with D-serine as substrate in presence and absence of 80 mmol/l {alpha}-aminoisobutyric acid (AIB). Nos. in brackets = no. of experiments.

 
Comicroinfusion of D-serine and AIB in late proximal rat nephrons. In a last set of experiments, we tested whether AIB has an inhibitory effect on D-serine reabsorption in the proximal tubule, thereby preventing D-serine nephrotoxicity. In earlier studies, we characterized D-serine reabsorption in rat kidney in vivo et situ in detail (39, 41). We found that D-serine is only reabsorbed in the S3 segment of the rat proximal tubule by a high-capacity/low-affinity transport system (Km {approx} 20 mmol/l). Therefore, in the present study, we microinfused D-[14C]serine (0.36 mmol/l) and [3H]inulin without or with 80 mmol/l AIB in late proximal nephrons and collected the final urine to measure the FR of the 14C activity. [Values of FR <100% indicate D-serine reabsorption specifically in the proximal straight tubule, since microinjection of D-serine in early distal tubule segments resulted in a FR of 100% (41).] Figure 9 shows that FR of D-[14C]serine in the final urine amounted to 44.6% and increased to 75.4% when 80 mmol/l AIB were present in the microinfusate. The latter FR value indicates a strong inhibitory effect of AIB on D-serine reabsorption, and its extent was similar to that found in previous work when 80 mmol/l D-[14C]serine alone was microinfused (41).


Figure 9
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  		Fig. 9. Fractional urinary recovery (%) of microinfused D-[14C]serine or [14C]AIB (0.36 mmol/l) in the presence and absence of 80 mmol/l comicroinfused AIB or D-serine during late proximal microinfusion in vivo et situ. Nos. in brackets = no. of experiments. Significant differences (P < 0.05) were found for column 1 vs. columns 2–6; column 4 vs. columns 5 and 6; and for column 5 vs. column 6.

 
To test to what extent D-serine influences the FR of AIB, we microinfused [14C]AIB (0.36 mmol/l) and [3H]inulin in the absence or presence of 80 mmol/l D-serine as well as 80 mmol/l AIB alone. [As with D-serine, AIB is not reabsorbed if microinjected in early distal tubule segments (unpublished observation).] As shown in Fig. 9, the FR of [14C]AIB rose from 30% to ~75% when 80 mmol/l D-serine was present and to ~60% when (in absence of D-serine) the [14C]AIB concentration was increased to 80 mmol/l. These results strongly suggest that D-serine and AIB compete for the same reabsorption mechanism.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Injection of high doses of D-serine [7.6 mmol (=800 mg)/kg body wt] in rats causes massive, generalized aminoaciduria, glucosuria, and proteinuria (6, 7, 22, 45) 4 h after administration. Our results are in accordance with earlier studies. Moreover, 2 h after D-serine administration (7.6 mmol/kg body wt), a mild hyperexcretion of all determined amino acids and glucose, except glutamine and phenylalanine, occurred. The time course of the FE indicates that the full extent of tubule damage has developed after 4 h.

To evaluate the dose-response relationship, we injected D-serine intraperitoneally at concentrations from 0.25 to 7.6 mmol (=26–800 mg)/kg body wt and measured the FE of amino acids and glucose 2 and 4 h later. Administration of the lowest dose of D-serine (0.25 mmol/kg body wt) showed no effect on the excretion of amino acids and glucose at either 2 or 4 h after administration. Plasma serine concentrations did not change significantly after this dose (control: 0.23 ± 0.02 mmol/l; 2 h: 0.25 ± 0.03 mmol/l; 4 h: 0.23 ± 0.06 mmol/l) in our model. Patients have been treated with nearly the same D-serine dose (16, 18, 43), i.e., 0.28 mmol·kg body wt–1·day–1. This treatment caused an increase in the D-serine plasma concentration from ~1.5 to 100–150 µmol/l (16). This led to a more than twofold increase in the total serine plasma concentration in these patients (16). This discrepancy between the human data and our data in rats might be explained by a more powerful D-serine oxidation in these animals than in humans. At a dosage of 0.76 mmol/kg body wt, D-serine led to a mild elevation of serine excretion 2 h after injection. However, the HPLC method used cannot distinguish between D- and L-isomers. Therefore, we do not interpret this mild hyperexcretion of serine as a D-serine-induced toxic effect. Rather, it seems most likely that the injected and filtered D-serine competes with the tubular reabsorption of endogenous D- and L-serine. As a consequence, the tubular transport capacity is exceeded, and serine is lost in the urine. After injection (4 h) of this dose, however, a slight hyperexcretion of aspartate, asparagine, glutamate, glycine, lysine, serine, and glucose became evident. We interpret this occurrence as a cytotoxic effect of D-serine. At this time, the serine plasma concentration had risen to 0.38 ± 0.05 mmol/l (control 0.23 ± 0.02 mmol/l).

The next higher D-serine doses of 2.54 and 7.6 mmol/kg body wt increased plasma serine to 0.54 ± 0.11 mmol/l and 1.01 ± 0.25 mmol/l, respectively, after 2 h and led to a mild aminoaciduria and glucosuria. (Figure 2 shows that the peak of the plasma concentration of D- and L-serine together was reached much earlier.) After administration of these doses (4 h), massive, generalized aminoaciduria and glucosuria had developed. By this time after the injection of 2.54 mmol (=260 mg)/kg body wt D-serine, at a plasma serine concentration of 0.36 ± 0.03 mmol/l, maximal tubule damage had occurred. Increasing the dose of D-serine to 7.6 mmol/kg body wt caused no further increase in FE of amino acids and glucose.

As mentioned above, the D-serine-induced tubulopathy is localized to the S3 segment of the proximal straight tubule. This is also the site where D-serine is reabsorbed via a low-affinity/high-capacity transport system (41). Because D-serine is not reabsorbed in the S1 and S2 segments (41), it is concentrated about threefold by water reabsorption along the proximal convoluted tubule before it arrives at its reabsorption site in the S3 segment. Thus, in this segment, there is a high D-serine concentration gradient between the tubule lumen and the intracellular compartment. Because this is the same tubule segment where the highest D-AAO activity has been found (25, 45), the exclusively late proximal localization of D-serine reabsorption (39, 41) optimizes the feeding of D-AAO with its substrate. Recently, Maekawa et al. (27) demonstrated in a rat model of functionally inactive renal D-AAO that D-serine is ineffective in causing tubulopathy when compared with control animals, thus indicating the importance of this enzyme for D-serine-induced nephrotoxicity.

We also tested the hypothesis that D-serine-induced hyperaminoaciduria is caused by oxidative stress of the tubule cells (see Fig. 1) and depletion of the tubular GSH content. We found indeed that D-serine treatment (7.6 mmol/kg body wt) halved the renal GSH concentration in the cortex and medulla. GSH treatment before and during D-serine injection not only prevented this decrease but also nearly normalized amino acid excretion. We therefore conclude that D-serine damages the straight proximal tubule because it is specifically reabsorbed there and subsequently metabolized by D-AAO to yield hydrogen peroxide. At high luminal D-serine concentrations, this metabolic process leads to a decrease in intracellular GSH and subsequently to oxidative destruction of the cells within several hours. Dispensing exogenous GSH before and during D-serine application keeps the cellular GSH content at normal levels and, at the same time, protects the S3 cells to such an extent that the severe D-serine-induced hyperaminoaciduria does not occur. This explanation, however, does not exclude the possibility that additional metabolic steps may play a role in D-serine-induced tubulopathy (47, 48).

AIB, a neutral, nonmetabolizable amino acid, is known to prevent tubular destruction in the S3 segment after coinjection with D-serine in equimolar doses (20, 21). We reexamined the nephroprotective AIB effect by measuring FE of amino acids and glucose. Indeed, the massive, generalized hyperaminoaciduria and glucosuria seen 4 h after injection of D-serine alone nearly disappeared in the presence of AIB. The mild hyperexcretion of some of the amino acids measured seems to indicate that AIB directly inhibits their reabsorption. The following observations support this view. Two groups of amino acids can be distinguished after coinjection of D-serine and AIB. Citrulline, glutamine, isoleucine, methionine, ornithine, and phenylalanine were not hyperexcreted after coinjection. In contrast, alanine, asparagine, glycine, lysine, and serine were mildly hyperexcreted after coinjection but not to the same massive extent as after D-serine injection alone. Exactly the same group of amino acids was also mildly hyperexcreted after injection of AIB alone. The FE of glucose remained at the control level after AIB injection only and after coinjection of AIB together with D-serine. These results indicate a full protection of S3 by AIB rather than a D-serine-induced toxic effect after coinjection of AIB and D-serine. Thus AIB induces a mild, specific aminoaciduria that can be explained by competition of AIB with the reabsorption of these amino acids.

These findings led to a consideration of the mechanism by which AIB produces its nephroprotective effect. One possibility considered was that AIB interferes with late-proximal D-serine reabsorption, thereby reducing D-serine uptake, leading secondarily to a smaller oxidation rate of D-serine and finally to a reduced generation of ROS. Therefore, we microinfused D-[14C]serine (and [3H]inulin) with or without 80 mmol/l AIB in late proximal nephrons and counted the radioactivity in the final urine. The results of our microinfusion experiments (see Fig. 9) show that the protective effect of AIB on D-serine nephrotoxicity can be explained at least partly by direct inhibition of cellular D-serine uptake by AIB. It is possible that AIB can also interfere with D-serine uptake at the basolateral cell side, but this is unknown.

A second mechanism by which AIB could possibly inhibit D-serine-induced tubulopathy is by interfering directly with D-AAO. We tested this possibility by determining D-AAO activity (with 8 mmol/l D-serine as substrate) in vitro in the absence and presence of 80 mmol/l AIB. AIB had no significant effect.

We therefore conclude that AIB inhibits D-serine-induced nephrotoxicity by reduction of D-serine uptake in the S3 segment rather than by direct inhibition of intracellular D-serine oxidation. As a consequence, less D-serine is available for intracellular D-AAO-mediated generation of ROS.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16294 and DeutscheForschungs gemeinschaft Grants Si 170/7-1 and SFB 176, TP A6.


   ACKNOWLEDGMENTS
 
We thank Olga Brokl for great encouragement and support during the time part of the present experiments were performed in Tucson at the Dept. of Physiology, University of Arizona.

Current address for A. W. Krug: Carl Gustav Carus University Hospital, Department of Medicine III, D-01307 Dresden, Germany.

Parts of this study have been presented as abstracts at the 29th annual meeting of the American Society of Nephrology, New Orleans, LA, in November 1996 (J Am Soc Nephrol 7: 1846, 1996) and at the 76th Congress of the Deutsche Physiologische Gesellschaft in Rostock, Germany, in March 1997 (Pflügers Arch 433: R37, 1997).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Silbernagl, Physiologisches Institut der, Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany (e-mail: stefan.silbernagl{at}mail.uni-wuerzburg.de)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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