Figure 1. The role of the kidney in total body amino acid homeostasis. This schematic representation of the main amino acid fluxes through the body shows that amino acids absorbed in the intestine reach the systemic circulation via the liver. Following a single meal the amount ingested may exceed the quantity found in the extracellular space by several fold. Therefore, the rapid cellular uptake and metabolic use of amino acids play central homeostatic roles. Liver, muscles, intestine, and kidney are major sites of amino acid metabolism. Additionally, to prevent their loss the kidneys reabsorb approximately 50 g/day of amino acids from the primary urine.

Figure 2. Scheme of nephron segments involved in amino acid reabsorption. Amino acids are freely filtered at the glomerulus and approximately 99.5% are reabsorbed in the proximal tubule (PT, blue color), mainly in the first and second segments (S1 and S2). Notable exceptions in humans are serine, glycine, histidine, and taurine, which lose a higher fraction in the urine.

Figure 3. Cellular model for the reabsorption of amino acids across a proximal tubule cells. Panel A shows a schematic representation of the luminal and basolateral amino acid transporters. Most amino acids are transported across the luminal membrane by symporters that use the electro‐chemical gradient of Na⁺ or H⁺ for driving the influx of amino acids. Neutral amino acids may be recycled to the lumen by the exchanger b^0,+AT‐rBAT (SLC7A9‐SLC3A1) to allow the uptake of cationic amino acids and cystine. The basolateral efflux of most nonessential neutral amino acids and of cationic amino acids is mediated by antiporters. Essential amino acids taken up in exchange for cationic amino acid efflux may recycle to the extracellular space via selective antiporters. Panel B depicts most of the known renal transport proteins; their protein and human gene names are indicated. In mice most of the transporters are expressed mainly in the early segments of the proximal tubule (S1 and S2). However, SIT1 (SLC6A20) is expressed along the entire proximal tubule, and B⁰AT3 (SLC6A18) and EAAT3 (SLC1A1), are expressed in the later proximal tubule (S2 and S3). Key: AA⁰ neutral amino acid, AA⁻ anionic amino acid, AA⁺ cationic amino acid, TMEM27 (collectrin).

Figure 4. Transport mechanisms: symporters, antiporters, and uniporters. Solutes are transported by secondary active transporters by an alternated access mechanism. The number of steps and solutes involved depends on the transport mechanism (uniporter, symporter, or antiporter). Transport mechanisms are classified based on the number of substrates simultaneously transported and the relative direction of substrate movements. (A and B) Alternative access mechanism for symporters and antiporters. (A) Symporters: Upon substrate (red circle) and ion (blue square with red center) binding to the open outward‐facing state (2), the substrate‐ion‐bound transporter changes to the occluded and inward‐facing states (172) releasing substrates. A transition between the unbound inward‐ and outward‐facing conformations is required to renew the transport cycle (4 and 1). For antiporters (B), substrate binding to the outward‐facing state (blue diamond) results in the transition to the inward‐facing states (172) and substrate release. Restoration of the outward‐facing state (4 to 1) requires the binding of a substrate to the inward‐facing transporter state (red circle) (5) to allow the transporter to return to the outward‐facing conformation and release the substrate (6). (C) Chart of each of transporter mechanism. Uniporters, move one solute along the solute concentration gradient; Symporters, translocate two or more substrates in the same direction, one solute (e.g., AA) eventually against its concentration gradient and the other solute (e.g., Na⁺) along its concentration gradient; Antiporters, translocate two substrates in opposite directions against the gradient concentration of one of the substrates. For symporters and antiporters, the presence of both substrates involved in the transport cycle is required for the translocation. All three mechanisms are utilitzed by amino acid and oligopeptide transporters. TAT1 (SLC16A10) is an example of an aromatic amino acid uniporter. B⁰AT1 (SLC6A19) is a symporter, cotranslocating neutral amino acids with sodium. PEPT1 (SLC15A1) is another symporter that cotransports peptides and protons. The heterodimeric amino acid transporter LAT2‐4F2hc (SLC7A8‐ SLC3A2) is an antiporter that exchanges neutral amino acids.

Figure 5. Chimeras and cysteine scanning experimental strategies to study the structure function relationship of solute transporters. Functional heterodimeric amino acid transporters (HATs) are formed by SLC7 transporters interacting covalently with accessory proteins of the SLC3 family, rBAT (SLC3A1) or 4F2hc (SLC3A2). To date rBAT has only been confirmed to interact with one SLC7 family member, b^0,+ (SLC7A9); while 4F2hc interacts with several members including LAT1 (Slc7a5). (A) Schematic representation of chimeras and truncations of rBAT and 4F2hc. To define the domain(s) of rBAT and 4F2hc involved in the recognition and interaction with the Slc7 transporters, chimeras, and truncations of rBAT and 4F2hc were constructed and in combination with b^0,+ and LAT1 functionally assayed. The chimeras were prepared by combining the following protein regions: cytoplasmic tail, transmembrane domain, neck, and glycosidase‐like domain. rBAT is represented in blue and 4F2hc in yellow. (B) L‐Arginine transport by b⁺⁰AT coexpressed with various chimeras. When coexpressed with b^0,+AT, chimeras containing rBAT cytoplasmic tail (BB44, BB4B) with or without the transmembrane domain (BBB4) transport L‐arginine indicating a role in the interaction between rBAT with b^0,+AT [adapted, with permission, from (70)]. (C) Schematic representation of targeted SERT residues. “Cysteine scanning” or the systematic introduction of cysteine or lysine mutations is another method for studying the relationship of specific residues to transporter function. To probe conformational changes occurring during the transport cycle the targeted residues are evaluated for cytoplasmic or extracellular accessibility and/or impact on transport function or protein expression. Rudnick and collaborators [adapted from reference (161) with permission] extensively studied the serotonine transporter (SERT/ SLC6A2), which is responsible for reuptake of 5‐hydroxytryptamine (5‐HT, Serotonin) in the postsynaptic membrane and is a target for antidepressants such as Fluoxetine (Prozac). The scheme for SERT is based on the LeuT model (discussed in the text). Residues studied by cysteine scanning are labeled in red. Combining the structural predictions based on the bacterial homologs and the cysteine scan, the authors suggested several possible protein conformations during the transport cycle that allow substrate binding and dissociation from both sides of the membrane.

Figure 6. The heterodimeric amino acid family. The HAT family is formed by glycoproteins from the SLC3 (rBAT and 4F2hc) family and amino acid transporters from the SLC7 family. (A) Chart of known SLC3 transporter partners. 4F2 interacts with several SLC7 transporters while rBAT is only known to interact with b^0,+AT. (B) Schematic representation of covalent interaction between SLC3 and SLC7 members. The SLC3 heavy subunits (pink), which are type II membrane glycoproteins with an intracellular NH2 terminus and a single transmembrane domain, and the SLC7 transporters [light subunit (blue)] are linked by a disulfide bridge (yellow) with conserved cysteine residues (e.g., cysteine 158 for the human xCT and cysteine 109 for human 4F2hc). [Adapted, with permission, from (140).]

Figure 7. Interaction of SLC6 family members with the Renin‐Angiotensin system proteins. (A) Schematic representation of B⁰AT1 interaction with RAS members ACE2 and TMEM27. ACE2 and TMEM27/Collectrin are type I integral membrane proteins with N‐terminal (N), transmembrane, and short C‐terminal (C) domains. ACE2 has one zinc‐binding motif (HEMGH) (bm) in the extracellular domain and is a carboxipeptidase with the consensus sequence P(Φ)_1‐3PΦ/+. The bradikinin‐(1‐8) peptide depicted (reversed) is one of the best in vivo targets of ACE2 (84). (B‐J) ACE2 and Tmem27/collectrin tissue specific colocalization with B⁰AT1. Representative immunofluorescent images of mouse tissue sections labeled with antibodies against B⁰AT1 (red) in small intestine (C) and kidney (E), and against Ace2 in small intestine (B) and Tmem27/collectrin in kidney (D). Panel (F) shows colocalization of B⁰AT1 and Ace2 in small intestine enterocytes (G) the colocalization of B⁰AT1 with Tmem27/collectrin in the proximal tubules. (H and I) Ace2 and Tmem27/collectrin knockout ablates B⁰AT1 expression in the small intestine or kidney, respectively. ACE2 and TMEM27 are encoded by X chromosome‐linked genes. Western blots of small intestine (H) and kidney (I) tissue lysates from wild‐type vs ace2^−/y and coll^−/y knockout mice probed with anti‐B⁰AT1 and anti‐β‐actin (loading control) antibodies (36,52). (J‐M) SIT1 colocalizes and functionally interacts with ACE2. SIT1 (SLC6A20) colocalizes with ACE2 in human small intestine (J, K, and L) and its coexpression with ACE2 in Xenopus laevis oocytes stimulates SIT1 transport (M).

Figure 8. Functional interaction between LAT2‐4F2 and TAT1. (A) Schematic representation of the functional interaction between LAT2‐4F2 and TAT1. LAT2‐4F2 recycles aromatic amino acids effluxed by TAT1 in exchange for efflux of neutral amino acids, which are not TAT1 substrates. (B) TAT1 does not physically associate with 4F2hc or LAT2. Coimmunoprecipitation was performed using lysates of biosynthetically ³⁵S‐met labeled oocytes coinjected with 4F2hc, LAT2, and TAT1 cRNAs (LT) or noninjected oocytes (NI) and analyzed by autoradiograph. Western blot (WB) of total lysates, or lysates immunoprecipitated using anti‐4F2 (IP‐4F2hc) antibodies detected LAT2 but not TAT1 coimmunopreciptated with 4F2hc. (C) Coexpression of TAT1 and LAT2‐4F2hc stimulates efflux of LAT2‐4F2 substrates that are not TAT1 substrates (e.g., L‐Gln). The amount of amino acids accumulating in the extracellular bath of oocytes with and without expression of LAT2‐4F2 and/or TAT1 was analyzed by UPLC [for details, see (150)]. The efflux of LAT2‐4F2 substrates (yellow) such as L‐glutamine, asparagine, serine and alanine was increased in the presence of TAT1. However, TAT1 (blue) substrate concentrations were not altered by coexpression of 4F2hc with TAT1. [Figure adapted from reference (150) with permission].

Figure 9. Expression of amino acid and oligopeptide transporters in kidney by region. (A) Scheme of proximal tubule. The proximal tubule segments extend from the kidney cortex to the outer medullary stripe (OM) of the medulla. The renal arcuate artery (red line) and vein (blue line) demarking the separation between the cortex and medulla are indicated. The glomerulus is indicated with (G). The proximal convoluted (S1) and (S2), and the proximal straight (S3) segments are separated with straight dotted lines. (B‐E) Reported expression of accessory proteins and transporters in proximal tubule segments S1 to S3. Panels B and C give expression data for apical, and panels D and E for basolateral localized proteins. (F) Scheme of distal nephron regions. The arcuate artery and veins separating cortex and medulla are indicated as in panel A, and the outer and inner medulla is separated by curved dotted lines. The thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct are indicated. (G and H) Reported expression of accessory proteins and transporters in distal nephron regions. The data shown for transporter and accessory protein expression in distal segments are incomplete and reflect cases in which the level of expression reported suggests a significant physiological role. No subcellular localization data are provided. Where no protein data are available, the mRNA expression is represented. For all panels (B‐E, G, and H) the data are represented using an arbitary scale of 0 to 100% expression—white being no reported expression with increasing expression indicated by darker colors. The relative expression of different genes is not represented. For all genes, a reference is given for the expression data represented.

Figure 10. Transporter basolateral versus apical membrane localization in kidney proximal tubule segments. (A‐I) Representative immunofluorescence images from labeled mouse kidney tissue sections of apical membrane amino acid transporters. Tissue sections were stained as follows: (A) B⁰AT1, (B) B⁰AT3, and (C) SIT1 are labeled in red, and in (A‐C) the basolateral membrane transporter, 4F2hc, is labeled with green (157). For panel (D), collectrin is labeled in green and shown without a labeled basolateral transporter (180). (E and F) The opposing axial distribution along proximal kidney tubule of mRNA for the HAT catalytic subunits, b^0,+AT, and rBAT, its glycoprotein partner. In situ hybridization of kidney section shown at a low magnification to demonstrate the individual HAT subunit mRNA gradients from cortex (c) to the medullary outer strip (os) and inner stripe (is) kidney regions (145). (G) Scheme of the apical versus basolateral localization of transporters expressed in kidney proximal tubules. Direction of transport and transporter mechanism is represented by arrows. Evidence exists for the expression of an as yet unidentified basolateral symporter and facilitative diffusion transporter(s) indicated as unlabeled transporters. (H‐I) Representative immunofluorescence images from stained mouse kidney tissue sections of basolateral localized amino acid transporters. The basolateral localized (H) SNAT3 (126) is labeled in red, (I) TAT1 (151) is labled in green, (J) LAT2 localization and (K) y⁺LAT1 transporters are labeled in red and (L) 4F2hc is labeled in green (126).

Figure 11. Renal amino acid metabolism in the proximal tubule. These diagrams illustrate some of the enzymes and transporters involved in the proximal tubule metabolism of Gln and Glu in panel A, and Arg, Cit, and NO in panel B. While the subcellular localization of proteins is indicated, the relative contributions of individual pathways to amino acid metabolism and transport is not shown (please refer to the text for more information). (A) Overview of L‐glutamine and L‐glutamate metabolic pathways and transporters in renal proximal tubule. Briefly L‐glutamine (Q) is taken up on the apical membrane by B⁰AT1 (SLC6A19) and B⁰AT3 (SLCA18) and L‐glutamate (E) is transported by EAAT3 (SLC1A1). Basolateral SNAT3 (SLC38A3) transports Q bidirectionally depending on the relative cytosolic versus extracellular Q concentration and pH. In the proximal tubule ammonia and ammonium (H⁺/NH₄ ⁺/NH₃,) are preferentially exported to the lumen by a number of systems. Here the apical NHE3 (SLC9A3) transporter is shown. NHE3 mediates H⁺ efflux, which may combine with NH₃ in the lumen to form NH₄ ⁺. NHE3 has also been reported to transport NH₄ ⁺. On the apical membrane Q of distal proximal tubule segments can be transaminated to E by γ‐glutamyl transferase (γ‐GT; EC 2.3.2.2). A number of SLC25 family transporters mediate mitochondrial transport of amino acids and metabolites. Internalized Q is taken up by unknown mitochondrial transporters where the mitochondrial phosphate‐dependent glutaminase (PDG; EC 3.5.1.2) converts Q to E and NH₄ ⁺. Recently, NH₄ ⁺ has been shown to be potentially transported by Aquaporin 8, which localized to the inner mitochondrial membrane. A further conversion of E to α‐KG and a NH₄ ⁺ can be catalyzed by glutamate dehydrogenase 1 (GDH1; EC 1.4.1.3) or E can be decarboxylated by glutamate decarboxylase (GAD; EC 4.1.1.15) to GABA and a bicarbonate ion (HCO₃ ⁻). Alternatively glutamic‐oxaloacetic transminase (GOT; EC 2.6.1.1) produces from E and pyruvate α‐KG and L‐aspartate (D). Mitochondrial TCA metabolism of α‐KG produces intermediates such as malate which can be exported to the cytoplasm for gluconeogeneisis. In the cytosol phosphenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) produces phosphoenolpyruvate (PEP) and CO₂ for gluconeogenesis and pyruvate formation. Finally, cytosolic glutamine synthetase (GS; E.C. 6.3.1.2) can convert E and NH₄ ⁺ to Q. (B) Proximal tubule transporters and enzymatic pathways in renal arginine metabolism. To summarize, apical b⁰⁺‐rBAT (SLC7A9‐SLC3A2) transporters uptake L‐arginine (R) in exchange for neutral amino acids (AA⁰), one source of AA⁰ is luminal B⁰AT1 (SLC6A19), which also takes up citrulline. On the basolateral membrane the anionic exchanger OAT1 (SLC22A6) is likely responsible for a large part of the citrulline uptake. The basolateral transporter TAT1 (SLC16A10) provides a facilitative diffusion pathway for aromatic AA⁰ which can be recycled by y⁺LAT1 (SLC7A7) in exchange for R efflux. Additionally, R may be exported by basolateral CAT1 (SLC7A1). In the proximal tubule cytoplasm, citrulline together with L‐aspartate (D) is converted to R and fumarate via the sequential action of arginosuccinate synthetase (ASS1; EC 6.3.4.5) and arginosuccinate lyase (ASL; EC 4.3.2.1). Nitric oxide synthase (NOS; EC 1.14.13.39) catalyses the production of nitric oxide (NO) and citrulline from R. Mitochondrial SLC25 family transport proteins exchange ornithine and R for citrulline. Within the mitorchondrial matrix ornithine can be converted to citrulline by ornithine transcarbamoylase (OTC: EC 2.1.3.3). Intramitochrondrial metabolism of R by arginine:glycine amidotransferase (AGAT; EC 2.1.4.1) produces ornithine and guanidinoacetate (GAA). R can also be decarboxylated by arginine decarboxylase (ADC; EC 4.1.19) to agmatine and CO_2. GAA can also be produced in the cytoplasm from R by cytosolic AGAT. GAA is released in the blood stream for creatine production. Mitochondrial argininase II (AII; EC 3.5.1.3) hydrolyzes R to produce ornithine. Transporters are indicated with ovals. Plasma membrane transporters, when known, are labeled with common and SLC names and the substrates translocation indicated with solid lines. Mitochondrial transporters are not labeled. Enyzmatic pathways are schematic and not all substrate intermediates are shown. Direct enzymatic pathways are shown with solid lines. Dotted lines indicate either translocation of substrates, for example, through the cytoplasm or extracellular space, or multiple metabolic steps. For more information regarding specific enzymes please refer to the Enzyme Commission (EC) numbers indicated in the figure legend.