Physiology and pathology of neutral amino acid transport in renal and intestinal epithelial cells

Abstract

About 95% of nutrient protein is absorbed in the mammalian intestine as amino acids and trl-and dipeptides. Once absorbed by the intestine amino acids are distributed by the circulation throughout the body. In the kidney an ultrafiltrate of the blood plasma is generated from which all amino acids are reabsorbed in the proximal tubule. System B{u2070} is the main amino acid transport system in these two tissues for broad neutral amino acids. Four transporters B{u2070}AT1 (SLC6A19), B{u2070}AT2 (SLC6A15), B{u2070}AT3 (SLC6A18) and ASTC2 (SCL1A5) have properties of this system when studied in isolation. B{u2070}ATl and B{u2070}AT2 co-transport one Na{u207A} -ion and one amino acid substrate. The low-affinity B{u2070}ATl transporter accepts most neutral amino acids, while the high-affinity transporter B{u2070}AT2 prefers branch-chained amino acids and proline. B{u2070}AT3 prefers alanine and glycine, but has also been reported to transport a variety of neutral amino acids. ASCT2 is a Na{u207A}-dependent antiporter, preferring neutral amino acids except those with an aromatic side-chain. However, in two studies it has been reported that amino acid transport of ASCT2 was inhibited also by phenylalanine. Analysis of ASCT2 function in vivo and in vitro, has led to the proposal that it represents the molecular correlate of system B{u2070} in kidney and intestine. Mutations in B{u2070}ATl, however, cause Hartnup disorder, a defect of neutral amino acid transport in kidney and intestine. Thus it is important to reconcile differences and to clarify the contribution of these amino acid transporters to system B{u2070}-like transport in kidney and intestine. Therefore, the overall aim for this thesis was to investigate the distribution and contribution of B{u2070}AT1 (SlC6A19), B{u2070}AT2 (SlC6A15), and ASTC2 (SCL1AS) to neutral amino acid transport in the kidney. Immunofluorescence studies revealed localisation of B{u2070}AT1 in the apical membrane of early (S1-S2) segments of the proximal tubule, while SOAT2 was localised to the apical membrane of the later (S2-S3) segments. This is consistent with physiological data reporting low-affinity transport in early segments and high-affinity transport in later segments of the proximal tubule. ASCT2 was localised in the apical membrane of S2 segments and in the basolateral membrane of the distal tubule. Transport studies with renal brush border membrane vesicles revealed a dominant transport activity that is consistent with the properties of B{u2070}AT1. This transport activity was lacking in B{u2070}AT1-deficient mice. B{u2070}AT2 activity was excluded by competition studies with selective substrates. ASCT2 activity was excluded due to the lack of additional amino acid transport in brush border membrane vesicles preloaded with A5CT2 amino acid substrates. Therefore, it is concluded that B{u2070}AT2 and ASCT2 do not contribute significantly to neutral amino acid transport in the kidney. B{u2070}AT1 is the main transport activity for neutral amino acid transport in the kidney. Show more