Orphan Transporter SLC6A18 Is Renal Neutral Amino Acid Transporter B⁰AT3^*

EXPERIMENTAL PROCEDURES

RESULTS

Since XT2 has been shown to be associated in kidney proximal tubule with Coll (5), we tested the possibility that XT2 would be expressed functionally in X. laevis oocytes in the presence of this associated protein. XT2 was therefore co-expressed in X. laevis oocytes with Coll and/or its homologue ACE2, and the uptake of l-isoleucine was tested. Unexpectedly, uptake of l-isoleucine was observed only when XT2 was co-expressed with ACE2, whereas no transport was observed when XT2 was expressed alone or together with Coll (Fig. 1A). Furthermore, co-expression of ACE2 and Coll with XT2 did not further increase the transport rate compared with XT2 plus ACE2.

The l-isoleucine transport induction upon ACE2 co-expression is accompanied by a slight increase of XT2 surface expression of 1.64 ± 0.39-fold when compared with XT2 alone (Fig. 1B). Furthermore, co-immunoprecipitation of ACE2 from oocyte membranes using XT2 antibody shows that ACE2 interacts with XT2 (Fig. 1C). These results suggest that ACE2 acts as a chaperone for XT2, slightly increasing its surface expression and more strongly its function in X. laevis oocytes. This co-expression renders the measurement of XT2 transport function possible in an expression system for the first time.

XT2 co-expression with ACE2 allowed the characterization of its transport selectivity, showing the transport of a broad range of neutral amino acids (Ala, Met, Val, Ile > Gly, Ser, Leu) (Fig. 2A). Concentration dependence experiments with l-Ile and Gly revealed a K_0.5 of XT2 for these substrates (0.21 and 0.53 mm, respectively) that is lower than that of B⁰AT1 (Fig. 2, B and C) (20). In contrast to B⁰AT1 but similarly to other members of the Slc6 family, such as the γ-aminobutyric acid transporter GAT1 (Slc6a1), the transport of l-Ile by XT2 is not only Na⁺- but also Cl⁻-dependent as well as pH-independent (Fig. 2, D and E). The Na⁺ dependence of Gly transport is sigmoidal, with a Hill coefficient that is higher than 1, suggesting that more than one sodium molecule is involved (data not shown). The precise ion transport stoichiometry of XT2 could not be investigated by two-electrode voltage clamp due to the low substrate transport rates.

To verify and examine the role of XT2 as an amino acid transporter in more detail, we reanalyzed the xt2 null mouse model (2) after more than 10 generations of backcrossing in C57BL/6 background. To evaluate whether the lack of XT2 induces compensatory changes, we examined the expression of the Slc6 family members B⁰AT1 and XT3 and of the associated proteins ACE2 and Coll at the mRNA and protein levels in xt2^−/− mice but observed no difference with wild-type littermate controls (supplemental Fig. 1, A and B).

To evaluate the impact of the loss of XT2 on the whole animal, we monitored food and water intake as well as urine and feces output in metabolic cages, but there were no differences in the measured parameters between the xt2^+/+ and xt2^−/− mice (supplemental Table 1). Analysis of plasma amino acids only suggested a slight but statistically not significant decrease of glycine and l-arginine in the xt2^−/− mice as compared with the xt2^+/+ mice (Fig. 3A). A massive loss of glycine in the urine of the xt2^−/− mice was confirmed, and the loss of other neutral amino acids, notably l-glutamine, was observed (Fig. 3B). To compare the reabsorption of amino acids, we estimated their fractional excretion. This is the proportion of the amino acids filtered by the kidney glomeruli that ends up in the urine (values of glomerular filtration rate were from Ref. 21). Whereas the fractional excretion of l-glutamine was only slightly augmented to 2.94% in xt2 null mice, those of glycine and l-methionine were strongly elevated to 51.37 and 33.41%, respectively (Table 1). These results show that despite aminoaciduria, these mice do not exhibit any compensatory mechanisms, implying that the normal diet readily provides excessive amounts of amino acids. The urinary peak corresponding to l-arginine unfortunately could not be distinguished from that of taurine, preventing the evaluation of l-arginine loss in xt2 null mice. Challenging xt2 null animals with a low protein diet (<0.5% crude protein) did not induce any additional difference in the metabolic cage parameters measured between xt2 null and wild-type mice (supplemental Table 1).

To assess the situation of the different partners of XT2 in vivo, we first checked the localization of XT2, Coll, and ACE2 in mouse kidney. As previously shown, XT2 localizes to the apical membrane in distal segments of the proximal tubule (S2, S3) and is not present in the initial S1 segment that begins at the glomerulus (3) (Fig. 4A). In contrast, ACE2 and Coll are both localized along the entire proximal tubule but with opposed gradients increasing toward the distal and proximal tubular ends, respectively (Fig. 4A and supplemental) Table 2. This shows that the main site of ACE2 expression coincides with the localization of XT2, suggesting that XT2 may interact with ACE2 in the proximal tubule in addition to its known interaction with Coll (5).

To test the role of these two potential associated proteins on XT2 expression in vivo, we used ace2 and coll null mice. At the mRNA level in the kidney, XT2 expression was unchanged in coll null mice, as shown previously (5), and in ace2 null mice (Fig. 4B). However, Western blot analysis of kidney brush border membrane proteins showed that XT2 was absent in the coll^−/y mice, in contrast to ace2^−/y and wild-type mice (Fig. 4, C and D). Immunofluorescence performed on kidney sections confirmed that XT2 is lacking in coll^−/y mice proximal tubule, whereas it is normally expressed in ace2^−/y as in wild-type control mice (Figs. 4, E and F). Amino acid fractional excretions of ace2^−/y and ace2^+/y mice were compared to assess the impact of ACE2 on XT2 function (Table 1). The results indicate that amino acid handling was not significantly different in the kidney of ace2 null mice compared with wild-type littermates. In contrast, aminoaciduria was very high in coll null mice, as reported previously (5). Note, however, that in these mice, several other amino acid transporters are affected that contribute to the aminoaciduria. These results confirm that the partner of XT2 in mouse kidney is indeed Coll and not ACE2, despite the fact that the expression gradient of Coll along the proximal tubule is opposed to that of both XT2 and ACE2 and that Coll unlike ACE2 does not allow XT2 function in X. laevis oocytes.

In the original study, xt2 null mice displayed, compared with wild-type littermates, a surprising elevation of blood pressure, as measured by tail cuff manometry. This difference was then abolished when drinking water was supplemented with glycine (0.1 g/ml) (2). The blood pressure of xt2 null mice was now retested using mice backcrossed more than 10 times into C57BL/6 background and using telemetry (Datasciences). The measurements showed no differences in blood pressure between the xt2^−/− and xt2^+/+ mice during the light phase when mice were not active (Fig. 5A). Similar results were obtained during the light and dark phases, whether the mice were active or not (data not shown). To test whether the previously measured increase in blood pressure was provoked by the stress of the tail cuff manometry method, telemetry measurements were repeated while physically restraining the mice. Submitting the animals to this stress provoked a higher mean blood pressure in xt2^−/− mice than in wild-type littermates (130.8 ± 0.89 versus 125.3 ± 1.40 mm Hg) and also a higher respiratory rate (Fig. 5B). This suggests an increased stress-induced reaction possibly involving a dysregulation of the sympathetic nervous system in xt2^−/− mice.

DISCUSSION

The first aim of this study was to directly characterize the function of the orphan transporter XT2 in the X. laevis oocyte expression system using its in vivo associated protein Coll. However, not Coll but only its homologue ACE2 supported the functional expression of XT2 in X. laevis oocytes such that its transport function of neutral amino acids expected from its homology with B⁰AT1 could be measured. This raised the suggestion that ACE2 might interact with XT2 in mouse kidney in addition to Coll. However, our experiments with the ace2 and coll null mice confirmed that the in vivo partner of XT2 is Coll and not ACE2. Indeed, XT2 expression and localization is normal in the proximal kidney tubule of ace2 null mice and absent in coll null mice. Furthermore, the absence of ACE2 does not affect the reabsorption of amino acids, whereas that of Coll leads to a strong aminoaciduria that is due to the lack of expression of XT2 and of other proximal tubule amino acid transporters (5). These observations suggest that during evolution, Coll took over the role of ACE2 in amino acid transport due to the increasing involvement of the latter as a key player in the renin-angiotensin system. It is, however, not clear why the restriction of functional XT2 association with ACE2 or Coll is just opposite in X. laevis oocytes as compared with the in vivo situation in kidney. Surprisingly, it appears that the difference observed in X. laevis oocytes is species-specific. Indeed, as suggested to us,⁵ co-expression of the human Coll ortholog with mouse XT2 generates an uptake similar to that with mouse ACE2 (data not shown). Further experiments will be required to identify potential additional cell-specific players that account for these differences.

The functional expression of XT2 together with ACE2 in oocytes allowed the long sought characterization of the function of this orphan transporter. Interestingly, its broad transport selectivity for neutral amino acids is clearly different from that of B⁰AT1. Particularly, whereas β-branched amino acids are very good substrates for XT2, as is the case for B⁰AT1, aromatic amino acids are apparently less efficiently transported. Importantly, l-Ala, l-Met, Gly, l-Ser, and l-Cys are also efficiently transported.

In contrast to B⁰AT1 that displays a Na⁺-dependent electrogenic neutral amino acid transport, XT2 is shown here to display a transport mode that is not only Na⁺-dependent but also Cl⁻-dependent with a K_0.5 for its preferred substrates that is lower than that of B⁰AT1. In that sense, the axial arrangement of XT2 that follows B⁰AT1 along the proximal tubule is similar to the sequential arrangement of low and high affinity glucose and peptide transporters SGLT2 and -1 and Pept1 and -2, respectively.

To correlate the measured in vitro function of XT2 with its in vivo function, we reanalyzed the xt2 null mouse model. Since the knock-out mice did not display compensatory changes of other amino acid transporters of the SLC6 cluster or of associated proteins, we hypothesize that the urinary phenotype of xt2 null mice is essentially due to the absence of XT2. In accordance with the in vitro results, we measured a broad aminoaciduria. Strikingly, there was not only a substantial loss of glycine (60-fold higher than in xt2^+/+) but also of l-glutamine (20-fold) and of all other neutral amino acids (Fig. 3). XT2 thus appears to be required for reabsorbing the leftover tubular amino acids that have not been reabsorbed in the early proximal kidney tubule segments by B⁰AT1 or the amino acids that would eventually backleak into the late proximal tubule segments through the paracellular route (22). Additionally, XT2 might also be required for the reabsorption of neutral amino acids that the luminal heterodimeric cystine and cationic amino acid transporter b^0,+AT-rBAT releases in exchange for its uptake substrates in the tubular lumen. Thereby, XT2 would interfere with l-arginine metabolism and nitric oxide synthesis (23). This latter hypothesis could be an explanation for the slightly decreased l-arginine plasma level and the tendency for elevated blood pressure observed in xt2 null mice.

Interestingly, XT2 had originally been identified in rat kidney as a renal osmotic stress-induced transcript (24). However, the experiments that we performed in mice could not verify such a regulation.⁶ This is either due to a species difference or to the less drastic conditions that we used, which might not have been sufficient to trigger this regulation.

In their original description, xt2 null mice displayed a significantly elevated blood pressure (2). This was, however, not observed in the present study after backcrossing these mice 10 times in a C57BL/6 background and using a telemetry system for the blood pressure measurements. Previous reports have already evidenced discrepancies between tail cuff manometry and telemetric blood pressure measurements and also highlighted the importance of the genetic background. In particular, the original xt2 null mouse had an Sv129 genetic background, a strain that contains two renin genes and the renal blood flow of which was suggested to be more sensitive toward nitric oxide than that of C57BL/6 mice (25). We could, however, observe in the present study a stress-sensitive hypertension and a stress-induced increase in respiratory rate in xt2 null mice, suggesting a possible impact of the lack of this transporter on regulatory functions of the sympathetic nervous system. Interestingly, plasma l-arginine of xt2^−/− mice was slightly lower than in the xt2^+/+ mice, and l-arginine is the precursor of nitric oxide, which, when released by endothelial cells, leads to vasodilation. Slightly lower circulating and renal levels of l-arginine might therefore induce susceptibility to an increase in blood pressure (26).

The role of XT2 in human seems to be limited since SLC6A18 has not been found associated with iminoglycinuria.⁷ Furthermore, a frequent single-nucleotide polymorphism that leads to a stop codon in SLC6A18 has not been linked to increased blood pressure (4). However, it is not excluded that the individual variability in stress-induced blood pressure increase could be linked to the function of this renal neutral amino acid transporter.

Supplementary Material