Regulation of renal amino acid transporters during metabolic acidosis

【摘要】  The kidney plays a major role in acid-base homeostasis by adapting the excretion of acid equivalents to dietary intake and metabolism. Urinary acid excretion is mediated by the secretion of protons and titratable acids, particularly ammonia. NH 3 is synthesized in proximal tubule cells from glutamine taken up via specific amino acid transporters. We tested whether kidney amino acid transporters are regulated in mice in which metabolic acidosis was induced with NH 4 Cl. Blood gas and urine analysis confirmed metabolic acidosis. Real-time RT-PCR was performed to quantify the mRNAs of 16 amino acid transporters. The mRNA of phosphoenolpyruvate carboxykinase (PEPCK) was quantified as positive control for the regulation and that of GAPDH, as internal standard. In acidosis, the mRNA of kidney system N amino acid transporter SNAT3 (SLC38A3/SN1) showed a strong induction similar to that of PEPCK, whereas all other tested mRNAs encoding glutamine or glutamate transporters were unchanged or reduced in abundance. At the protein level, Western blotting and immunohistochemistry demonstrated an increased abundance of SNAT3 and reduced expression of the basolateral cationic amino acid/neutral amino acid exchanger subunit y + -LAT1 (SLC7A7). SNAT3 was localized to the basolateral membrane of the late proximal tubule S3 segment in control animals, whereas its expression was extended to the earlier S2 segment of the proximal tubule during acidosis. Our results suggest that the selective regulation of SNAT3 and y + LAT1 expression may serve a major role in the renal adaptation to acid secretion and thus for systemic acid-base balance.

【关键词】  ammoniagenesis amino acid transporter acidbase

THE KIDNEY PLAYS A CENTRAL role in maintaining systemic pH and in the defense against systemic disturbances of pH and bicarbonate concentrations ( 18 ). Under normal conditions, the kidneys reabsorb filtered bicarbonate as well as regenerate bicarbonate for buffering nonvolatile acid equivalents stemming from metabolism. The process of regeneration occurs through the synthesis of ammonia, which results in the production of new bicarbonate and the excretion of protons ( 18, 29 ). In addition, ammonia is needed as urinary buffer for secreted protons along the collecting duct. There, H + secretion by the vacuolar H + -ATPases creates a steep proton gradient across the apical membrane of intercalated cells. This proton gradient opposes further H + excretion unless protons are buffered in urine by so-called titratable acids (TA) ( 50, 51 ). The major TA comprise phosphate, citrate, and NH 3 ( 19 ). Thus renal ammonia synthesis serves three major tasks in the control of systemic acid-base balance: 1 ) the regeneration of HCO 3 -, 2 ) the excretion of protons in the form of NH 4 + from the proximal tubule, and 3 ) the buffering of protons in the collecting duct as TA supporting ongoing H + excretion by vacuolar H + -ATPases ( 29 ).

NH 3 /NH 4 + is synthetized in the proximal tubule from glutamine taken up from urine and blood involving several intermediate steps. One molecule of ammonium (NH 4 + ) is generated through the action of the phosphate-dependent glutaminase (PDG) leaving one glutamate. Glutamate is further metabolized, either producing -ketoglutarate and one more NH 4 +, leading eventually through the pathways of gluconeogenesis and tricarboxylic acid cycle to two molecules, HCO 3 - and glucose (or CO 2 ), or resulting in the generation of one molecule of HCO 3 - and pyruvate ( 29 ). Thus, depending on the metabolic pathway, one molecule of glutamine can result at maximum in the generation of two molecules of NH 4 + and two HCO 3 - ions. During metabolic acidosis, glutamine is released from liver and muscle, and the renal extraction of glutamine as well as its metabolism in kidney is stimulated and favored in the direction of gluconeogenesis ( 11, 52 ). Stimulation of ammonium synthesis involves the upregulation of several enzymes in the proximal tubule, including the phosphate-dependent glutaminase and cytosolic phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in gluconeogenesis ( 13, 15, 29, 30 ). Furthermore, glutamine uptake into proximal tubular cells is stimulated ( 55, 56 ). It is less clear whether uptake of glutamate via the luminal glutamate transporter EAAC1 (SLC1A1) also contributes to ammoniagenesis as indicated by in vitro data ( 27 ). Basic ammonium synthesis in the proximal tubule occurs under normal conditions mainly in the late proximal tubule (S3 segment) ( 28, 29, 44 ), whereas during metabolic acidosis, ammonium synthesis and excretion also are found in earlier segments of the proximal tubule ( 29 ). Glutamine uptake into the cells of the proximal tubule can occur both on the apical side from primary urine across the brush-border membrane and on the basolateral side from blood involving several amino acid transport systems ( 40 ). However, glutamine is almost completely removed from urine along the proximal tubule ( 40 ), and thus increased glutamine delivery for ammonium synthesis depends on uptake from blood across the basolateral membrane. In agreement, several studies have shown that glutamine transport is increased in basolateral membrane vesicles prepared from acidotic animals ( 29, 55, 56 ). This glutamine transport is Na + dependent, and it was recently suggested to be mediated by the system N amino acid transporter SNAT3 (SLC38A3), which is upregulated during acidosis ( 41 ).

Over the past decade, a large number of amino acid transport systems have been identified on a molecular level, allowing examination of their functional characteristics, regulation, and physiological role (for review, see Refs. 7, 10, 32, 46, 47, 49, 57 ). Renal amino acid transporters serve a variety of different functions. A major group of transporters serves mainly in the reabsorption of freely filtered amino acids, either mediating their cellular uptake across the luminal membrane or mediating their release at the basolateral side into interstitium and blood. Some transporters accepting osmolytes like betaine or taurine are involved in the release and uptake of these osmolytes, which are important for the survival of cells in the medulla, where large changes in osmolarity occur ( 40 ). Another group of transporters may be mainly involved in the housekeeping function, providing renal cells with amino acids for basal metabolism. Accordingly, amino acid transporters have to fulfill different and highly specialized tasks and should also be differentially localized and regulated ( 7 ).

Members of several amino acid transporter subfamilies have been shown to transport glutamine and to be expressed in the kidney ( 5 ). However, little is known about which transporters are regulated during acidosis and may thereby contribute to renal metabolism and ammoniagenesis. Therefore, we used a mouse model for metabolic acidosis (NH 4 Cl in the drinking water) and real-time RT-PCR to examine the regulation of renal amino acid transporters that are accepting glutamine or glutamate as substrates and are regulated under these conditions. We have confirmed that SLC38A3 (SN1, SNAT3), a Na + -dependent glutamine transporter, is highly upregulated on mRNA and protein levels. We localized this transporter by immunohistochemistry to the basolateral membrane of the S3 segment of the proximal tubule under control conditions and also found its expression in earlier segments during acidosis. In addition, we found that the catalytic subunit of the basolateral system y + L amino acid transporter y + -LAT1 was downregulated. All other transporters were not affected by acidosis. These results suggest that SLC38A3 may be important for the basolateral uptake of glutamine and that a concerted upregulation of its expression, together with the downregulation of an amino acid exit pathway, may both contribute during acidosis to the increased availability of glutamine for ammonium synthesis, particularly in the earlier segments of the proximal tubule.

MATERIALS AND METHODS

Animal Studies

NMRI mice (Charles River Laboratories, Sulzfeld, Germany) (male, 12 wk, 35-45 g) were maintained on standard chow and had access to drinking water ad libitum. To induce metabolic acidosis, mice were given 2% sucrose-0.28 M NH 4 Cl in the drinking water for 48 h or 7 days as described previously ( 2, 42 ). Each group consisted of four animals for each time point and treatment, and experiments were repeated three times, respectively. For blood analysis, mice were anesthetized with ketamine-xylazine, and heparinized mixed arterial-venous blood samples were collected and analyzed immediately for blood gases and electrolytes on a Radiometer ABL 505 blood gas analyzer (Radiometer, Copenhagen, Denmark). Urine was collected as spot urine, and pH was immediately measured using a pH microelectrode (Lazar Research Laboratories, Los Angeles, CA) connected to a Thermo Orion 290 pH meter. Creatinine and ammonia were measured using the Jaffe and Berthelot protocols, respectively ( 37 ). All animal studies were approved by the Swiss Kantonales Veterinäramt, Zurich.

Real-Time RT-PCR

RNA extraction and reverse transcription. Mice were killed by intraperitoneal injection of ketamine-xylazine and subsequent cervical dislocation. Kidneys were rapidly harvested and frozen immediately until further use. Total mRNA was extracted from 30 mg of kidney tissue by using the RNA Aqueous 4PCR kit (Ambion) according to the manufacturer's instructions. For RNA extraction, kidney tissue was thawed in RNALater solution (Ambion), transferred to lysis buffer, and homogenized on ice with an Elvehjem potter. RNA was bound on columns and treated with DNase for 15 min at 30°C temperature to reduce genomic DNA contamination. Quantity and purity of total eluted RNA were assessed by spectrometry and on agarose gels. Each RNA sample was diluted to 200 ng/µl, and 1 µl was used as template for reverse transcription with the TaqMan reverse transcription kit (Applied Biosystems).

For reverse transcription, 200 ng of RNA template were diluted in a 20-µl reaction mix that contained (final concentrations) RT buffer (1 x ), MgCl 2 (5.5 mM), random hexamers (2.5 µM), RNase inhibitor (0.4 U/µl), the multiscribe reverse transcriptase enzyme (1.25 U/µl), dNTP mix (500 µM each), and RNase-free water.

Real-time PCR. Real-time PCR was performed as described previously ( 14 ) according to the recommendations supplied by Applied Biosystems ( http://home.appliedbiosystems.com ). Primers for all genes of interest were designed using Primer Express software from Applied Biosystems (see Table 1 ). Primers were chosen to result in amplicons of 70-150 bp that span intron-exon boundaries to exclude genomic DNA contamination. The specificity of all primers was first tested on mRNA derived from kidney, liver, and brain and always resulted in a single product of the expected size (data not shown). Probes were labeled with the reporter dye FAM at the 5' end and with the quencher dye TAMRA at the 3' end (Microsynth, Balgach, Switzerland). The passive reference dye (ROX) was included in the TaqMan buffer supplied by the manufacturer. cDNA (20 µl) obtained from the RT reaction was diluted to 100 µl with RNase-free water. A 25-µl PCR reaction volume was prepared by using 5 µl of diluted cDNA as template with sense and antisense primers (25 µM each) and the labeled probe (5 µM). The TaqMan Universal PCR Master Mix (Applied Biosystems) was added to the final volume. Prior quantification experiments had determined the optimal concentrations of primers and probes and the temperature settings to yield maximal fluorescence signals and PCR products. Reactions were run in 96-well optical reaction plates with the use of a Prism 7700 cycler (Applied Biosystems). Thermal cycles were set at 95°C (10 min) followed by 40 cycles at 95°C (15 s) and 60°C (1 min) with auto ramp time. For analyzing the data, the threshold was set to 0.06, because this value had been determined to be in the linear range of the amplification curves for all mRNAs in all experimental runs. All reactions were run in duplicate. The abundance of the target mRNAs was calculated relative to a reference mRNA (GAPDH). Since standard curves made for all primer pairs on kidney or brain RNA had revealed an efficiency value close to 2 (degree of increase in input mRNA required to decrease the cycle number by 1), relative expression ratios were calculated as R = 2 [Ct(GAPDH) - Ct(test)], where Ct is the cycle number at the threshold and test stands for the tested mRNA.

Table 1. Primers and probes used for real-time PCR

Western Blotting

Mice were killed, and kidneys were rapidly harvested and used for either preparation of total crude membranes or isolation of brush-border membrane vesicles (BBMV). BBMV were prepared using the Mg 2+ precipitation technique as described previously ( 4 ).

Total crude membranes were prepared by homogenization of total kidneys in ice-cold K-HEPES buffer (200 mM mannitol, 80 mM K-HEPES, 41 mM KOH, pH 7.5), with pepstatin, leupeptin, K-EDTA, and PMSF added as protease inhibitors, with a tip sonicator. Samples were centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was saved. Subsequently, the supernatant was centrifuged at 100,000 g for 1 h at 4°C, and the resultant pellet was resuspended in K-HEPES buffer containing protease inhibitors. After measurement of the total protein concentration (Bio-Rad protein kit), 50 µg of crude membrane protein or 10 µg of BBMV protein were solubilized in Laemmli sample buffer, and SDS-PAGE was performed on 10 and 12% polyacrylamide gels. For immunoblotting, proteins were transferred electrophoretically from unstained gels to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). After blocking with 5% milk powder in Tris-buffered saline-0.1% Tween 20 for 60 min, the blots were incubated with the primary antibodies and mouse monoclonal anti-actin (42 kDa; Sigma) at 1: 5,000 either for 2 h at room temperature or overnight at 4°C. The primary antibodies were rabbit anti-mouse LAT-2 (serum 560, 1:2,000) ( 38 ), rabbit anti-mouse y + -LAT-1 (affinity-purified antibody 398, 1:500) ( 3 ), rabbit anti-mouse b 0,+ AT (sera 400 and 558, 1: 2,000) ( 35 ), goat anti-rabbit 4F2hc (200 µg/ml, 1:1,000; Santa Cruz Biotechnology), rabbit anti-EAAC1 (affinity purified, 1:1,000; Alpha Diagnostics International, San Antonio, TX), rabbit anti-NHE3 1:10,000 (kind gift of Dr. O. Moe, University of Texas, Dallas, TX), and two different rabbit anti-SNAT3 antibodies. One antibody was directed against a NH 2 -terminal peptide (NH 2 -MEIPRQTEMVELVPNGKC-CONH 2 ) derived from the rat sequence. The peptide was linked to keyhole limpet hemocyanin, rabbits were immunized (Pineda Antibody Service, Berlin, Germany), and the serum was affinity purified (Sulfolink kit 44895; Pierce Biotechnology). The second antibody has been described previously ( 17 ). After washing and subsequent blocking, blots were incubated with secondary antibodies conjugated with alkaline phosphatase (goat anti-rabbit, 1: 5,000, and donkey anti-goat, 1:5,000; Promega, Madison, WI), for 1 h at room temperature. Antibody binding was detected using the CDP Star kit (Roche Diagnostics, Indianapolis, IN) before exposure to X-ray film (Kodak) or by taking images with the Diana III chemiluminescence detection system (Raytest). Protein quantification was done using AIDA Image analyzer version 3.44. The specificity of the antibodies used was tested by preincubation with the immunizing peptides and preimmune sera (data not shown). Data from Western blotting are expressed relative to actin, which was tested on the same membranes after stripping and reprobing. The mean of the control was taken as 100%, and values for the treated groups are expressed as percent change. Gauss's law of error propagation was used to calculate the standard error of the mean. Data were tested for significance using the unpaired Student's t -test and ANOVA, and only values with P < 0.05 were considered statistically significant.

Immunohistochemistry

Mice were anesthetized and perfused through the left ventricle with PBS followed by paraformaldehyde-lysine-periodate (PLP) fixative ( 25 ). Kidneys were removed, flushed with fixative solution, and post-fixed overnight at 4°C by immersion in PLP. Organs were washed three times with PBS, and 5-µm cryosections were cut after cryoprotection with 2.3 M sucrose in PBS for at least 12 h. Immunostaining was carried out as described previously ( 14, 48 ). Briefly, sections were incubated with 1% SDS for 5 min, washed three times with PBS, and incubated with PBS containing 1% bovine serum albumin for 15 min before addition of the primary antibody. The primary antibodies (see Western Blotting ) were diluted in PBS at dilutions of 1:20 to 1:1,000 and applied either for 75 min at room temperature or overnight at 4°C. Sections were then washed twice for 5 min with high-NaCl PBS (PBS + 2.7% NaCl) and once with PBS and then incubated with the secondary antibodies (donkey anti-rabbit, donkey anti-mouse, and donkey anti-goat at dilutions of 1:1,000, 1:400, and 1:400, respectively; Molecular Probes, Portland, OR) for 1 h at room temperature. Sections were again washed twice with high-NaCl PBS and once with PBS before mounting with VectaMount (Vector Laboratories, Burlingame, CA). Sections were viewed with a Leica SP1 UV CLSM confocal microscope. Pictures were processed and assembled using Adobe Photoshop. All antibodies were tested for their specificity by using preimmune sera and immunizing peptides. The secondary antibodies alone did not result in significant staining (data not shown).

RESULTS

Animal Model

To induce metabolic acidosis, animals were given 0.28 M NH 4 Cl-2% sucrose in the drinking water for 48 h or 7 days. A group receiving only 2% sucrose in the drinking water served as a control. In addition, a fourth group received 0.28 M NaHCO 3 -2% sucrose in the drinking water to induce mild metabolic alkalosis. These treatments have been shown to induce metabolic acidosis and alkalosis, respectively ( 42 ). To control for the effect of the diets on systemic acid-base status and urinary acidification, blood and urine were collected and analyzed ( Table 2 ). As expected, NH 4 Cl induced metabolic acidosis after 48 h and 7 days as evident from the reduction in venous blood pH and bicarbonate and serum chloride increased. Urinary pH was decreased under these conditions, indicating increased excretion of acid equivalents by the kidneys. Urinary ammonia/ammonium excretion was increased in acidotic animals as expected. In contrast, animals receiving NaHCO 3 for 7 days had higher serum bicarbonate levels and excreted more alkaline urine with low ammonia/ammonium, consistent with the induction of metabolic alkalosis.

Table 2. Blood gas and electrolytes and urine pH from control, acidotic, or alkalotic animals

Real-Time RT-PCR

To identify mRNAs regulated under these conditions, RNA was extracted from total kidneys where care was taken that in all samples, cortex and medulla were similarly represented by cutting coronal slices from whole kidneys. Real-time RT-PCR was performed for several control genes and genes of amino acid transporters known to be expressed in kidney and capable of glutamine or glutamate transport. These transporters included members of three distinct families: the SLC1 family of glutamate transporters ( 20 ), the SLC3 and SLC7 families of heteromeric amino acid transporter subunits ( 33, 45, 49 ), and the SLC38 family of system A and N glutamine transporters ( 24 ). The control genes included PEPCK, a key enzyme in the process of gluconeogenesis that has been shown to be induced by metabolic acidosis and other treatments stimulating renal ammoniagenesis ( 13, 15 ), the Cl - /anion exchanger pendrin (SLC26A4) ( 26 ), the Na + /H + exchanger NHE3 (SLC9A3) ( 31 ), and GAPDH. Pendrin has been previously shown to be downregulated by NH 4 Cl loading on both mRNA and protein levels ( 16, 34, 48 ). NHE3 is only regulated on the protein level, not mRNA ( 1 ), and GAPDH served as an internal standard for later normalization as described previously ( 14 ). The relative abundance of the mRNAs of the GAPDH and NHE3 control genes did not change significantly under all treatments ( Table 3 ). In contrast, induction of metabolic acidosis with NH 4 Cl resulted in a strong decrease in the relative mRNA abundance of pendrin after 2 and 7 days as expected ( Table 3 ). Also, NaHCO 3 in the drinking water resulted in a small decrease of pendrin mRNA. The abundance of PEPCK mRNA increased dramatically under NH 4 Cl treatment to 447.3 ± 12.5 and 268.0 ± 15.6% after 2 and 7 days, respectively, and was slightly but significantly reduced after NaHCO 3 to 72.3 ± 13.4% ( Table 3 ). Together, treatment of animals with NH 4 Cl or NaHCO 3 resulted in the expected changes in mRNA levels of pendrin and PEPCK, whereas the abundance of NHE3 and GAPDH mRNA was not altered in agreement with earlier studies ( 1 ).

Table 3. Summary of real-time PCR data

Real-time RT-PCR demonstrated that most of the amino acid transporters tested were expressed at the mRNA level in kidney as estimated from the Ct values. However, the Ct values of SLC38A4 and SLC38A5 36, indicating very low or no expression. Also, SLC38A1 had a Ct value of 35, suggesting very low abundance ( Table 3 ). The relative mRNA abundance of most amino acid transporters tested was not altered by loading the animals with either NH 4 Cl or NaHCO 3, as listed in Table 3. However, significant changes were found for the y + LAT1 (SLC7A7) subunit of system y + L, which was strongly reduced after 2 and 7 days of NH 4 Cl (to 28.4 ± 21.4 and 52.6 ± 21.3%, respectively; Table 3 ). Similarly, the relative abundance of the b 0,+ AT (SLC7A9) mRNA was strongly decreased ( Table 3 ). In contrast, the mRNA of one member of the SLC38 family of system A and N amino acid transporters was increased by NH 4 Cl, confirming similar results in rats ( 41 ). The abundance of SLC38A3 (SNAT3) mRNA was already high under control conditions (30.3 ± 0.2 cycles), and its relative abundance increased to 608.9 ± 18.0% after 2 days and to 416.3 ± 16.9% after 7 days of NH 4 Cl, suggesting a strong stimulation of its expression ( Table 3 ). Induction of metabolic alkalosis by NaHCO 3 loading had no significant effect on SLC38A1 or SLC38A3 mRNA abundance. The only mRNA showing a significant increase in its relative abundance was SLC38A2, to 214.5 ± 14.5% of control after 7 days of NaHCO 3 loading ( Table 3 ). Together, these results suggest that metabolic acidosis is associated with specific changes in the relative mRNA abundance of three amino acid transporters increasing SLC38A3 (SNAT3) and decreasing SLC7A7 (y + LAT1) and SLC7A9 (b 0,+ AT) mRNA.

Western Blotting

Western blotting from crude membrane fractions prepared from total kidney of control and treated animals was used to test for the effects on protein abundance of amino acid transporters and on pendrin and NHE3 as control proteins. Metabolic acidosis resulted in decreased expression levels of pendrin and increased expression levels of NHE3, confirming the effect of NH 4 Cl loading on protein level ( Fig. 1 ).

Fig. 1. Effect of acidosis on the Na + /H + exchanger NHE3 and pendrin protein abundance. Immunoblotting was performed on a total kidney crude membrane fraction to confirm the effect of NH 4 Cl and NaHCO 3 loadings on the protein levels of the control proteins NHE3 and pendrin (d, days). The expression levels of NHE3 were increased during acidosis and decreased during alkalosis as reported previously ( 1 ), whereas the protein abundance of pendrin was decreased during NH 4 Cl loading ( 48 ).

Similar to the findings on mRNA level, treatment with NH 4 Cl or NaHCO 3 did not significantly alter the abundance of several amino acid transporters tested. In particular, the abundance of b 0,+ AT was not altered despite the massive downregulation on mRNA level ( Fig. 2 ). Also, the expression of the luminal glutamate transporter EAAC1 (SLC1A1) was not changed in either total membrane preparations or an isolated brush-border membrane fraction ( Fig. 2 ). However, the protein abundance of SLC38A3 was severalfold increased (327.6 ± 55.1% and 904.1 ± 212.2%, respectively) after 2 and 7 days of NH 4 Cl, in agreement with the strong increase found on mRNA level ( Fig. 3 ). No change was observed with alkalosis (data not shown). Also, consistent with the real-time PCR data, y + -LAT1 protein abundance decreased with NH 4 Cl loading to 47.3 ± 8.8% ( Fig. 4 ), whereas the other basolateral members of the SLC7 family, i.e., 4F2hc and LAT2, were not altered. Thus metabolic acidosis caused increased protein expression of the system N amino acid transporter SNAT3 (SLC38A3) and reduced expression of the y + LAT1 subunit of the heteromeric amino acid transporter family.

Fig. 2. Acidosis does not alter SLC7A9 (b 0,+ AT) and SLC1A1 (EAAC1) protein abundance. A : despite a massive reduction in SLC7A9 mRNA during NH 4 Cl loading, no change in the corresponding protein levels could be observed in a total kidney membrane fraction. B : to test for a possible accumulation of EAAC1 in the luminal brush-border membrane, brush-border membrane vesicles were prepared and tested for EAAC1 and -actin abundance. Neither acidosis nor alkalosis altered the relative abundance of EAAC1 in the brush-border membrane.

Fig. 3. Acidosis increases SNAT3 (SLC38A3) protein expression in the kidney. Induction of acidosis with NH 4 Cl loading increased the protein abundance of the glutamine transporter SNAT3 in the total kidney membrane fraction. Bar graph presents intensity of SNAT3 bands normalized against actin staining.

Fig. 4. Acidosis selectively decreases y + LAT1 (SLC7A7) abundance. The expression levels of the basolateral y + LAT1 subunit were strongly decreased during acidosis, whereas the abundance of the 4F2hc (SLC3A2) and LAT2 (SLC7A8) was not affected. In contrast, 7 days of alkalosis had no impact on y + LAT1 abundance.

Immunohistochemistry

Immunohistochemistry was used to examine the localization of the system N transporter SNAT3 (SLC38A3) in kidney and to test whether acidosis altered the segmental distribution of the 4F2, y + LAT1, b 0,+ AT, and LAT2 subunits and the EAAC1 glutamate transporter. The subcellular localization of SNAT3 was recently reported by Solbu et al. ( 41 ) and was shown to be restricted to the basolateral side of late proximal tubules. In contrast, Karinch et al. ( 21 ) had suggested on the basis of Western blot data that SNAT3 may be expressed in both apical and basolateral membranes of rat kidney proximal tubules. Immunostaining with affinity-purified antibodies against SNAT3 demonstrated strong labeling of late proximal tubules (S3 segments) with exclusive staining of basolateral membranes ( Fig. 5 ). Brush-border staining was never observed. To confirm the localization of SNAT3 in the late proximal tubule, double labeling was performed using the 4F2hc subunit, which localizes mainly to basolateral membranes of the earlier segments of the proximal tubule (S1 and S2 segments) ( 38 ). Antibodies against 4F2hc strongly labeled proximal tubule segments that were negative for SNAT3 staining, but more juxtamedullary proximal segments (late proximal tubule, S3) stained strongly for SNAT3. Thus, in mouse kidney from control animals, SNAT3 is strongly expressed in the basolateral membrane of late proximal tubules.

Fig. 5. The system N amino acid transporter SNAT3 is localized to the basolateral side of late proximal tubules under control conditions. Immunostaining of mouse kidneys was performed with affinity-purified antibodies against SNAT3 (red) and the 4F2hc subunit (green), which is mainly expressed at the basolateral side of early proximal tubules. A : both SNAT3 and 4F2hc stained basolateral membranes of proximal tubules; however, little overlap was observed, demonstrating localization of SNAT3 in the late proximal tubule. B : higher magnification of a late proximal tubule (S3 segment) stained with antibodies against SNAT3 showing labeling of the basolateral membrane with multiple invaginations. Original magnifications, x 400 and x 630, respectively.

In kidneys from mice treated with NH 4 Cl for 2 and 7 days, the intensity of staining for SNAT3 was increased, consistent with the higher abundance observed by Western blotting. Moreover, staining also was observed in the earlier S2 segment of the proximal tubule ( Fig. 6 ).

Fig. 6. Acidosis induces expression of SNAT3 in early proximal tubule. Immunostaining for SNAT3 was performed in mouse kidneys from control animals and animals loaded with NH 4 Cl for 7 days. Top : in kidneys from control mice, SNAT3 staining was only observed in late proximal tubules in the outer stripe (OS) of the outer medulla and medullary rays ( left ), whereas in kidneys from acidotic mice, SNAT3 staining also was strong in the cortex (C), indicative of expression in early proximal tubules ( right ). No staining was observed in the inner stripe (IS) of the outer medulla under any condition. Bottom : colocalization (merge) of 4F2hc (green) and SNAT3 (red) show overlap in kidneys from acidotic mice due to expression of SNAT3 in earlier segments of the proximal tubule (S2 segments).

Double-staining of mouse kidney with antibodies against 4F2 and y + LAT1 demonstrated that the segmental localization of these transporters subunits was not distinguishable in control kidneys and tissue from animals treated with either NH 4 Cl or NaHCO 3. The most prominent labeling was seen in the basolateral membrane of early proximal tubules (S1 segment) with decreasing intensity toward the end of the proximal tubule (S3 segment) as described previously ( 3 ). Also, the subcellular localization was not different. However, the intensity of staining for y + LAT1 was reduced in kidneys obtained from animals with 2 or 7 days of NH 4 Cl, consistent with reduced protein abundance ( Fig. 7 ). Similarly, immunostaining for the b 0, + AT transporter subunit and the glutamate transporter EAAC1 was seen in the brush-border membrane of the proximal tubule as described previously ( 35, 39 ), and no difference in subcellular localization and segmental distribution was found (data not shown).

Fig. 7. Effect of acidosis on y + LAT1 and 4F2hc distribution and intensity of staining. Staining against y + LAT1 (red) and its subunit 4F2hc (green) was observed at the basolateral side of early proximal tubules in control kidneys as described previously ( 3 ). In kidneys from acidotic animals (7 days of NH 4 Cl), the intensity of staining for y + LAT1 was dramatically reduced (pictures were taken with the same settings of the laser scanning microscope), whereas the staining for 4F2hc was not changed. The segmental distribution of y + LAT1 and 4F2hc was affected by acidosis. G glomerulum, S1 early segment of the proximal tubule. Original magnification, x 500.

DISCUSSION

To identify amino acid transporters that may be involved in providing the proximal tubule with glutamine for ammoniagenesis, we used an animal model for metabolic acidosis and screened amino acid transporters known to be expressed in kidney and transporting glutamine. Pendrin, PEPCK, and NHE3 served as control genes that confirmed the effect of NH 4 Cl loading and metabolic acidosis together with blood gas and urine analysis. Our screen revealed that only three amino acid transporter genes were significantly regulated by metabolic acidosis. The mRNA abundance of the y + LAT1 (SLC7A7) subunit of the basolateral system y + L exchange transporter was strongly decreased. Surprisingly, the abundance of the catalytic subunit b 0,+ AT of the major luminal transport system for cationic amino acids and cystine was also decreased. In contrast, b 0,+ AT protein abundance remained unchanged, whereas the protein abundance of y + LAT1 was reduced. Interestingly, the abundance of the heavy chain subunit 4F2hc was not altered. This may be explained by the fact that 4F2hc serves as a subunit for several basolateral transport systems [i.e., system L (LAT2)] and that a downregulation would affect all heteromeric basolateral amino acid transport systems (see also Fig. 8 ). Thus a specific downregulation of system y + L occurs through the modification of y + LAT1 mRNA and protein abundance. System y + L serves as an efflux pathway for cationic amino acids such as arginine and lysine in exchange for neutral amino acids and Na + ( 36, 43 ). A downregulation of this pathway could lead to accumulation of these amino acids in the proximal tubule cells, where at least arginine could fuel into ammoniagenesis and gluconeogenesis and thus contribute to pH regulation.

Fig. 8. Cell model of proximal tubule cell with amino acid transporters. Scheme shows epithelial amino acid transporters localized in proximal tubule cells and their contribution to transcellular amino acid transport and potential role in fuelling ammoniagenesis. Amino acid transporter names are given according to the HUGO nomenclature. AA +, cationic amino acids; AA 0, neutral amino acids; CssC, cysteine; PDG, phosphate-dependent glutaminase; GDH, glutamate dehydrogenase; PEPCK, cytosolic phosphoenolpyruvate carboxykinase; KG, -ketoglutarate.

The second amino acid transporter identified as being strongly regulated during metabolic acidosis is SNAT3 (SLC38A3). SNAT3 mRNA and protein abundance was severalfold increased during metabolic acidosis in parallel with changes in PEPCK mRNA abundance. This observation is in agreement with an earlier observation in rat kidney ( 21, 41 ). Immunohistochemistry demonstrated that SNAT3 is expressed on the basolateral membrane of the straight part of the proximal tubule (S3 segment) and thus positioned to serve as an import pathway for glutamine. In the kidneys from animals treated with NH 4 Cl, SNAT3 expression also was observed in the earlier S2 segment of the proximal tubule. However, in contrast to a recent report that SNAT3 protein had been observed in a brush-border membrane fraction, no signal for SNAT3 expression in the brush-border membrane of the proximal tubule could be observed. In view of the highly polarized expression of transporters in the kidney proximal tubule and the supposed physiological role of SNAT3, it is very unlikely that SNAT3 would be expressed in the apical membrane. Glutamine uptake in proximal tubule cells is highly stimulated during metabolic acidosis and requires that enough glutamine is supplied. However, glutamine levels in urine are constant, and virtually all glutamine is reabsorbed during the passage of the primary urine through the proximal tubule ( 40 ). Thus uptake of glutamine from urine would not be sufficient under conditions of metabolic acidosis. On the other hand, the glutamine concentration in blood is high, and during metabolic acidosis it is even higher due to the stimulation of glutamine release from liver and muscle ( 52 ). Uptake of glutamine from blood across the basolateral membrane would provide a large enough pool of glutamine ( Fig. 8 ). The basolateral glutamine uptake into proximal tubule cells is Na + dependent ( 55, 56 ), and SNAT3 has been characterized in several heterologous expression systems as a Na + -dependent transporter for glutamine, asparagine, and histidine ( 6, 8 ). Glutamine transport by SNAT3 also is associated with proton antiport ( 9 ); however, it has been debated whether proton transport is coupled or uncoupled ( 6, 8 ). Although extracellular pH influences SNAT3 transport activity, the direction of glutamine transport is mainly determined by the glutamine gradient ( 6, 8 ). This gradient is clearly inwardly directed in the proximal tubule because of the rapid intracellular metabolism of glutamine and the high blood concentrations of glutamine. Thus SNAT3 shows the functional characteristics, regulation, and localization of the basolateral glutamine transporter providing glutamine for renal ammonia synthesis. Along this line, we also observed that the recently identified apical system B 0 Na + -dependent neutral amino acid transporters B 0 AT1 (SLC6A19) and SIT1 (SLC6A20) ( 22, 23 ) are not affected by acidosis or alkalosis (Romeo E, Makrides V, Wagner CA, Verrey F, unpublished results).

Interestingly, we did not find any evidence for regulation of the apical glutamate transporter EAAC1 (SLC1A3) that has been implicated in the adaptive response. Glutamate uptake into the porcine proximal tubule-like cell line LLC-PK 1 is stimulated by chronic acidification of the culture medium ( 27 ). However, in vivo, the activity of the brush-border membrane glutamate-producing ectoenzyme phosphate-independent glutaminase is decreased during acidosis, and apical glutamate entry into proximal tubular cells is rather reduced, serving as a stimulus for higher glutamine fluxes through the intracellular phosphate-dependent glutaminase pathway ( 54 ).

During metabolic acidosis, the kidney increases proton secretion through a concerted stimulation of several mechanisms contributing to ammoniagenesis and ammonia excretion. Several enzymes such as the phosphate-dependent glutaminase and PEPCK increase the utilization of glutamine and the subsequent synthesis of NH 3 and HCO 3 -; in parallel, glutamine synthesis from other amino acids via the glutamine synthetase is reduced, at least in mouse kidney ( 12 ), and the excretion of NH 3 /NH 4 + across the apical membrane via the NHE3 exchanger is stimulated ( 1 ). In this context, upregulation of SNAT3 provides the necessary uptake step for glutamine, underlying the efficient extraction of glutamine from renal blood as documented in different species ( 52, 53 ). A cell model of the proximal tubule depicts the various amino acid transporters tested in this study and the possible interaction with ammoniagenesis ( Fig. 8 ).

In summary, metabolic acidosis in mice is associated with the selective regulation of two renal amino acid transporters expressed in the proximal tubule. Whereas the abundance of the y + LAT1 subunit of the basolateral cationic amino acids efflux pathway is reduced, the expression of the basolateral Na + -coupled glutamine transporter SNAT3 is highly stimulated. Immunohistochemistry localized SNAT3 to the basolateral membrane of late proximal tubules with expression also in earlier segments during metabolic acidosis. SNAT3 may therefore be an important regulator for renal ammoniagenesis and eventually for systemic acid-base homeostasis. The parallel regulation of y + LAT1 and SNAT3 suggests also that both transport proteins may be part of concerted regulatory mechanisms aiming at increasing intracellular amino acid concentrations for ammonia synthesis and subsequent excretion via apical Na + /H + exchangers, i.e., NHE3.

GRANTS

This study was supported by Swiss National Research Foundation Grants 31-108021/1 (to F. Verrey) and 3100B0-109677/1 (to C. A. Wagner), a grant from the Theodor and Ida Herzog-Eggli Foundation, Zurich (to F. Verrey and C. A. Wagner), the European Union 6th Framework EUGINDAT grant (to F. Verrey and C. A. Wagner), University Research Priority Program "Integrative Human Physiology" of the University of Zurich (to C. A. Wagner), and Welch Foundation Grant AQ-1507 (to J. X. Jiang).

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作者单位：1 Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland; and 2 Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas