Renal amino acid transport systems and essential hypertension

The FASEB Journal article fj.12-224998. Published online April 24, 2013.

The FASEB Journal • Review

Renal Amino acid Transport Systems and Essential Hypertension Vanda Pinto,*,† Maria João Pinho,* and Patrício Soares-da-Silva*,1 *Department of Pharmacology and Therapeutics, Faculty of Medicine, and †Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal Several clinical and animal studies suggest that “blood pressure goes with the kidney,” that is, a normotensive recipient of a kidney genetically programmed for hypertension will develop hypertension. Intrarenal dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport. The candidate transport systems for L-DOPA, the source for dopamine, include the sodiumdependent systems B0, B0,ⴙ, and yⴙL, and the sodiumindependent systems L (LAT1 and LAT2) and b0,ⴙ. Renal LAT2 is overexpressed in the prehypertensive spontaneously hypertensive rat (SHR), which might contribute to enhanced L-DOPA uptake in the proximal tubule and increased dopamine production, as an attempt to overcome the defect in D1 receptor function. On the other hand, it has been recently reported that impaired arginine transport contributes to low renal nitric oxide bioavailability observed in the SHR renal medulla. Here we review the importance of renal amino acid transporters in the kidney and highlight pathophysiological changes in the expression and regulation of these transporters in essential hypertension. The study of the regulation of renal amino acid transporters may help to define the underlying mechanisms predisposing individuals to an increased risk for development of hypertension.— Pinto, V., Pinho, M. J., Soares-da-Silva, P. Renal amino acid transport systems and essential hypertension. FASEB J. 27, 000 – 000 (2013). www.fasebj.org

ABSTRACT

Key Words: kidney 䡠 dopamine 䡠 nitric oxide 䡠 blood pressure Hypertension, or high blood pressure, is a major contributor to cardiovascular risk. It has been recently reported that ⬃25% of the adult population worldwide

Abbreviations: 4F2hc, 4F2 heavy chain; AAT, amino acid transporter; CAT, cationic amino acid transporter; COMT, catechol-O-methyl-transferase; DOPAC, 3,4-dihydroxyphenylacetic acid; L-AADC, l-amino acid decarboxylase; L-DOPA, l-dihydroxyphenylalanine; LAT1, L-type amino acid transporter type 1; LAT2, L-type amino acid transporter type 2; L-NAME, N-G-nitro-l-arginine methyl ester; MAO, monoamine oxidase; MBF, medullary blood flow; MLDF, medullary laser Doppler flux; NHE3, Na⫹,H⫹-exchanger 3; NO, nitric oxide; NOS, nitric oxide synthase; PTE, proximal tubular epithelial; SD, Sprague-Dawley; SHR, spontaneously hypertensive rat; SS, Dahl salt-sensitive; WKY, Wistar Kyoto 0892-6638/13/0027-0001 © FASEB

suffers from hypertension (1). As many as 90 –95% of all cases of hypertension are classified as essential (primary) hypertension with unknown cause of the disease (2). The remaining 5–10% of cases (secondary hypertension) are caused by known factors, including endocrine disorders, kidney diseases, and tumors (3). This severe condition is associated with a significant increase in the risk for progression to heart failure, arrhythmias, or sudden death (4). The causes that underlie hypertension are complex, because both genetic and environmental factors participate in the pathogenesis of this medical condition. It has been estimated, however, that 30 –50% of essential hypertension is heritable. Approximately 30 –35% of subjects with normal blood pressure are salt sensitive; in patients with hypertension, this percentage is as high as 50 –70% (5). The kidney is important in the long-term regulation of blood pressure and is the major organ involved in the regulation of sodium homoeostasis. In spontaneously hypertensive rats (SHRs) of the Okamoto-Aoki strain, Dahl salt-sensitive (SS) rats, Milan hypertensive rats, and Prague hypertensive rats, arterial hypertension can be transferred with a renal graft from the hypertensive strain to normotensive recipients. Furthermore, renal grafts from the respective normotensive control strains lowered arterial pressure in these genetically hypertensive rat strains (6 –9). Many studies have focused on the abnormal renal handling of salt in the pathogenesis of hypertension (10, 11). As discussed below, the majority of amino acid transporters (AATs) that are dysregulated in hypertension are associated with the availability of renal dopamine and nitric oxide (NO). Therefore, this review will emphasize the renal dopaminergic system and the relationship between NO and renal medullary blood flow (MBF).

RENAL DOPAMINERGIC SYSTEM In the mammalian kidney, dopamine is primarily produced in the proximal tubule (12). The dopamine 1 Correspondence: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal. E-mail: [email protected] doi: 10.1096/fj.12-224998

1

precursor l-dihydroxyphenylalanine (L-DOPA) is filtered at the glomerulus and is taken up by the proximal tubule, where it is converted to dopamine by aromatic l-amino acid decarboxylase (L-AADC) (13). The regulation of this non-neuronal dopaminergic system depends mainly on the availability of L-DOPA, on its decarboxylation into dopamine and on cell outward amine transfer mechanisms (13, 14). L-DOPA uptake in renal epithelial cells is promoted through the sodiumindependent and pH-sensitive L-type AAT type 2 (LAT2), the activity of which might rate-limit the synthesis of renal dopamine. As depicted in Fig. 1, renal dopamine is metabolized predominantly by catechol-Omethyl-transferase (COMT) and monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetic acid (DOPAC), and to homovallinic acid (13, 14). The effects of dopamine, in mammals, are mediated by 5 dopamine receptor subtypes. These 5 receptor subtypes differ in their primary structures and show distinct affinities for dopamine receptor agonists and antagonists (15). The D1-like receptors are composed of the D1 and D5 receptor subtypes (D1A and D1B, for rodent homologues). The D1-like receptors couple to the stimulatory G proteins Gs and Gq and activate adenylate cyclase activity to increase cytosolic cAMP levels (11). The D2-like receptors are composed of the D2, D3, and D4 receptor subtypes, which couple to the inhibitory G proteins Gi and Go and modulate ion channel activity and/or inhibit adenylate cyclase activity. All of the dopamine receptor subtypes have been shown to regulate, directly or indirectly, sodium transport in the proximal and distal nephron and blood pressure (11, 16). The mechanisms through which renal dopamine is

*

L-DOPA

DA

Na+

H+

D1R

D1R

NHE3

Apical

L-DOPA AADC

DA 3MT ATP

ADP K+

DOPAC HVA

D1R

NKA

Basolateral Na+

*

DA

Figure 1. Hypothetical scheme of dopamine synthesis in the proximal tubles of rat kidney. 3MT, 3-methoxytyramine; AADC, l-aromatic amino acid decarboxylase; COMT, catechol-O-methyl-transferase; HVA, homovanillic acid; L-DOPA, L-dihydroxyphenylalanine; MAO, monoamine oxidase; NHE3, Na⫹,H⫹-exchanger 3; NKA, Na⫹,K⫹-ATPase. Asterisks indicate inhibition of NHE3 and NKA. 2

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thought to produce natriuresis involve the activation of D1-like receptors that inhibit the activity of 2 final effector proteins, the Na⫹,H⫹-exchanger 3 (NHE3) and the Na⫹,K⫹-ATPase (17). Several clinical observations, as well as studies in animals with various forms of genetic hypertension, suggest that defects in the renal dopaminergic system may contribute to the development of hypertension by causing salt retention (11). The SHR has been extensively used to elucidate the mechanisms of the defective D1-like receptor function in proximal tubules. Dopamine production in SHRs is normal or even increased, but the natriuretic effect of dopamine is diminished as a result of a defective transduction of the D1 receptor signal (18). Defective D1-like receptor function has also been reported in proximal tubules of SS rats (19). SS rats produce renal dopamine but have a poor natriuretic and diuretic response to a high-salt diet (20).

MEDULLARY BLOOD FLOW AND NO Studies have shown that reductions in MBF can have a profound effect on the long-term control of arterial pressure in genetic rat models of hypertension. It is recognized that that 15–30% reductions of blood flow to the renal medulla can lead to the development of hypertension (21). There is considerable evidence that NO production within the renal medulla plays a major role in the regulation of MBF. Nitric oxide plays an important role in regulation of vascular tone, renal sodium handling, and systemic blood pressure. Macula densa NO production influences the tubuloglomerular feedback control of afferent arteriolar resistance, thereby enhancing autoregulation of renal blood flow and renin secretion (22). Chronic intravenous administration of the NO synthase (NOS) inhibitor N-G-nitro-l-arginine methyl ester (L-NAME), at a dose that produced no change in cortical blood flow, resulted in a sustained 30% reduction of MBF and chronic hypertension (23). Moreover, chronic intramedullary infusions of L-NAME into a single remaining kidney of Sprague-Dawley (SD) rats resulted in a 30% reduction of MBF with no change of cortical blood flow, resulting in sodium retention and hypertension (24). This suggested that dysfunction in NO production in the renal medulla can have an effect on the entire cardiovascular system. In SS rats, an increase of daily NaCl intake (from 0.4 to 4.0% of diet) produced a 30% reduction of MBF over the first 24 to 48 h after switching to the high-salt diet. Salt-induced hypertension was prevented by the chronic intramedullary infusion of l-arginine, the substrate for NOS (25).

RENAL EPITHELIAL AATS AATs are responsible for the uptake of amino acids derived from diet in the small intestine, the release into

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TABLE 1. Epithelial amino acid transport systems and their properties System

A ASC asc B0 B0,⫹ b0,⫹ ␤ Gly IMINO L

N PAT T ⫺ XAG x⫺ c y⫹ y⫹L

Protein

SLC

Substrates

Ion dependence

SNAT2 SNAT4 ASCT1 ASCT2 4F2hc/asc1 B0AT1 B0AT2 ATB0,⫹ rBAT/b0,AT ␶T B0AT3 IMINO 4F2hc/LAT1 4F2hc/LAT2 LAT3 LAT4 SNAT3 SNAT5 PAT1 PAT2 TAT1 EAAT2 EAAT3 4F2 hc/xCT CAT-1 4F2hc/y⫹LAT1 4F2hc/y⫹LAT2

SLC38A2 SLC38A4 SLC1A4 SLC1A5 SLC3A2/SLC7A10 SLC6A19 SLC6A15 SLC6A14 SLC3A1/SLC7A9 SLC6A6 SLC6A18 SLC6A20 SLC3A2/SLC7A5 SLC3A2/SLC7A8 SLC43A1 SLC43A2 SLC38A3 SLC38A5 SLC36A1 SLC36A2 SLC16A10 SLC1A2 SLC1A1 SLC3A2/SLC7A11 SLC7A1 SLC3A2/SLC7A7 SLC3A2/SLC7A6

G,P,A,S,C,Q,N,H,M G,A,S,C,Q,N,M A,S,C A,S,C,T,Q G,A,S,C,T AA0 P,L,V,I,M AA0,AA⫹ R,K,O,Cn Tau G P H,M,L,I,V,F,Y,W AA0 except P L,I,M,F L,I,M,F Q,N,H Q,N,H,S,G G,P,A G,P,A F,Y,W E,D E,D D,E,Cn R,K,O,H K,R,Q,H,M,L K,R,Q,H,M,L,A,C

Na⫹ Na⫹ Na⫹ Na⫹ Na⫹ Na⫹ Na⫹,Cl⫺ Na⫹,Cl⫺ Na⫹,Cl Na⫹,Cl

Na⫹(S),H⫹(A) Na⫹(S),H⫹(A) H⫹ H⫹ Na⫹,H⫹(S), K⫹(A) Na⫹,H⫹(S), K⫹(A) Na⫹,(S-AA0) Na⫹,(S-AA0)

Amino acids are given in 1-letter codes. A, antiport; AA0, neutral amino acids; AA⫹, cationic amino acids; Cn, cystine; O, ornithine; S, symport; S-AA0, symport together with neutral amino acid.

the blood, and subsequent uptake of amino acids from the blood into tissues or the reabsorption of amino acids from the urine along the kidney nephron. In the central nervous system, AATs regulate the transport of amino acids across the blood-brain barrier or are involved in the reuptake of neurotransmitter amino acids such as glycine, aspartate, or glutamate from the synaptic cleft and are important for the metabolic coupling of astrocytes and neurons. Other AATs are involved in basic cellular functions such as cell volume regulation, the synthesis of glutathione, the provision of amino acids for protein synthesis and energy metabolism. In the kidney, the proximal tubule is the major site of nutrient reabsorption. About 95–99% of all amino acids are reabsorbed in the proximal convoluted tubule and proximal straight tubule. All kidney cells express some AATs that are involved mostly in house-keeping functions. Some kidney cells also require additional uptake of amino acids, which are used as precursors for the synthesis of paracrine and/or endocrine substances such as NO (26). As reviewed previously, 5 transport activities in kidney and intestine have been proposed (Table 1 and ref. 27): the neutral system, transporting all neutral amino acids; the basic system, transporting cationic amino acids together with cysteine; the acidic system, transporting glutamate and aspartate; the iminoglycine system, transporting proline, hydroxyproline, and glycine; and the ␤-amino acid system. These AMINO ACID TRANSPORTERS IN HYPERTENSION

transport activities are based on functional studies in kidney and intestine and the amino acid profile in the urine of individuals with different aminoacidurias (Table 2). Transporter dysfunction has also been reported in a wide spectrum of primary human cancers. Tumor cells up-regulate of a set of AATs to support tumor metabolism and trigger cell growth and survival, as discussed below. TABLE 2. Disorders associated with altered epithelial amino acid transport systems Protein

SLC

ASCT2

SLC1A5

B0AT1

SLC6A19

rBAT/b0,AT B0AT3 IMINO 4F2hc/LAT1

SLC3A1/SLC7A9 SLC6A18 SLC6A20 SLC3A2/SLC7A5

4F2hc/LAT2 PAT2 EAAT3

SLC3A2/SLC7A8 SLC36A2 SLC1A1

CAT-1 4F2hc/y⫹LAT1

SLC7A1 SLC3A2/SLC7A7

Disorder

Tumor growth, hypertension Hartnup disorder, hypertension Cystinuria Hypertension? Iminoglycinuria Tumor growth, hypertension Hypertension Iminoglycinuria Dicarboxylic aminoaciduria Hypertension Lysinuric protein intolerance

3

B0AT1 (SLC6A19) and B0AT3 (SLC6A18)

rBAT/b0,ⴙAT (SLC3A1/SLC7A9)

Neutral amino acids represent ⬎80% of the free plasma amino acids and are all transported by the luminal B0AT1 transporter (SLC6A19), though with different apparent affinities (26, 28). This neutral amino acid cotransporter with broad selectivity is expressed in the luminal brush border membrane of the early segments of the kidney proximal tubule and similarly along the small intestine (29). Expressed in Xenopus oocytes, the B0AT1 cDNA induces a sodiumdependent, chloride-independent uptake of neutral amino acids with an affinity for l-leucine uptake (28). Its defect has been shown to cause Hartnup disorder, an autosomal recessive condition which is characterized by a urinary loss of neutral amino acids (30). B0AT3 (SLC6A18) is highly expressed in the apical membrane of the proximal tubules in the kidney. Functionally, B0AT3 transports a broad range of neutral amino acids with high affinity in a sodium- and chloride-dependent manner (31) and appears to be required for the reabsorption of tubular amino acids leftover by B0AT1 in the early proximal kidney tubule segments or amino acids that have leaked back through the paracellular pathway (32). Some AATs have been shown to interact with other proteins for appropriate localization and function. This interaction guarantees the insertion of the transporter in the membrane and may modulate the activity or the supply of substrate. In the mouse proximal tubule the expression of B0AT1, as well as that of B0AT3 and IMINO (see below), have been shown to depend on the expression of collectrin (TMEM27), a short type I transmembrane protein (25 kDa) that is ⬃40% identical with the membrane anchor region of the reninangiotensin system enzyme ACE2 (33). B0AT1 and B0AT3 were strongly down-regulated in the kidneys of TMEM27-knockout mice, which led to massive amounts of amino acids lost in the urine (34). In contrast to other partner proteins, such as 4F2 heavy chain (4F2hc) and rBAT, the association between TMEM27 and B0AT1/ B0AT3 is thought to occur through noncovalent interactions (35).

Cationic amino acids and the disulfide-linked l-cysteine dimer enter the epithelial cells via the cystinuria transporter (b0,⫹AT). This transporter is made of a catalytic subunit belonging to the SLC7 family and a disulfidelinked accessory subunit referred to as heavy chain and called rBAT (SLC3A1; ref. 27). Similarly to the basolateral heterodimeric exchangers of the SLC7/SLC3 family, it functions as an obligatory antiporter and specifically exchanges its cationic substrates or l-cystine against intracellular neutral amino acids. b0,⫹AT mRNA is expressed in kidney and small intestine and, to a smaller extent, in heart, liver, placenta, and lung. rBAT protein was found in the apical membrane of renal proximal tubules, increasing from the S1 to the S3 segment and in the microvilli of the small intestine. On the other hand, although b0,⫹AT protein is also expressed in the apical membrane of the proximal tubule, expression levels decrease from the S1 to the S3 segment (38).

IMINO (SLC6A20) Another member of the same SLC6 AAT cluster, SLC6A20 in humans, has been identified as the molecular correlate of system IMINO, mediating the cotransport of l-proline (36, 37). This transporter, called SIT1 or IMINO, is localized to the brush border membrane of the proximal tubule in mice (29). As expected for system IMINO, it also transports hydroxy-l-proline and betaine but does not transport glycine. IMINO appears to require, as B0AT1 and B0AT3, the association with collectrin for its surface expression in renal tubules. This is suggested due to the low brush-border membrane expression of IMINO in collectrin-knockout mice and by the large urinary l-proline loss in these animals (34). 4

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EAAT3 (SLC1A1) The high-affinity transporter for anionic amino acids, referred to as EAAT3 (SLC1A1), is expressed in the intestine and kidney, as well as in the brain (39, 40). It is localized in the proximal tubule brush-border membrane with an axial gradient: low amounts in S1 and highest levels in the later segments S2 and S3 (40). This transporter has been shown to cotransport its substrates with 3 sodium ions and 1 proton in exchange for 1 potassium ion (41). Unlike the SLC6 and SLC7 transporters, no SLC1-associated transmembrane protein has been identified. 4F2hc/LAT1 (SLC3A2/SLC7A5) The best-characterized basolateral transporters of the proximal tubule amino acid reabsorption machinery function as obligatory exchangers (system L and system y⫹L) and thus do not perform net basolateral amino acid export (42). System L conveys the sodium-independent transport of large branched and aromatic neutral amino acids in almost all types of cells (38). The first isoform of system L, LAT1 (SLC7A5), preferentially mediates the sodium-independent transport of large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine, and is a major route for providing tumor cells with branched and aromatic amino acids (43). LAT1 is widely expressed in nonepithelial cells, such as brain, spleen, thymus, testis, skin, liver, placenta, skeletal muscle, and stomach and has a high affinity for amino acid substrates (43, 44). Recent studies have demonstrated that LAT1 is a major L-type AAT in a variety of cancer cells, including hepatic, oral, breast, bladder, and colon (45). Although the transport of leucine by LAT1 in LLC-PK1 porcine renal cells has been previously described (46), LAT1 has a very limited tissue distribution in the kidney (47). The heavy sub-

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unit 4F2hc (SLC3A2) brings LAT1 to the plasma membrane. In the absence of 4F2hc, LAT1 is found in intracellular compartments, whereas 4F2hc can reach the plasma membrane independently (48). Moreover, 4F2hc interacts with several light chains to form system L (with LAT1 and LAT2; Fig. 2), system y⫹L (with y⫹LAT1 and y⫹LAT2), system x⫺ c (with xCT), or system asc (with asc1) (38). 4F2hc/LAT2 (SLC3A2/SLC7A8) LAT2 (SLC7A8) is highly expressed in polarized epithelia (49), suggesting an important role in transepithelial amino acid transport, but it has a lower affinity for amino acid substrates than LAT1 (38, 49). LAT2 is a major sodium-independent AAT, and its functionality is dependent on the abundance of 4F2hc. LAT2 also transports small neutral amino acids, such as l-alanine, l-glycine, l-cysteine, l-serine, and glutamine, all of which are poor substrates for LAT1 (50). 4F2hc/yⴙLAT1 (SLC3A2/SLC7A7) System y⫹L was first functionally described in erythrocytes (51). However, its presence has also been revealed in placenta, liver, small intestine, and kidney (52–55). It has an axial distribution along the kidney proximal tubule and small intestine similar to that of LAT2 (56, 57). y⫹LAT1 (SLC7A7) plays an important role for the transepithelial transport of its cationic substrates, which it transports out of the cells in exchange for extracellular neutral amino acids and sodium (58, 59). The functional importance of this electroneutral exchange for transcellular cationic amino acid transport is demonstrated by the disease lysinuric protein intolerance, caused by mutations of the y⫹LAT1 gene which is AA0

ASCT2 (SLC1A5) ASCT2 is a sodium-dependent exchanger of neutral amino acids that belongs to the SLC1 family and has been shown to be expressed at the mRNA level in kidney. ASCT transporters are sodium-dependent obligatory exchangers of amino acids (in particular alanine, serine, cysteine, and threonine) that are structurally related to the EAAT transporters. ASCT2 belongs to a restricted group of transporters that share specificity for glutamine, since glutamine is the major precursor of urinary ammonia, thus playing a key role in acid-base homeostasis (63, 64). As for LAT1, ASCT2 is expressed in primary human cancers and several cancer cell lines, where it has been shown to play essential roles in growth and survival (65). Cationic AAT 1 (CAT-1) (SLC7A1) The inhibition of NOS with inactive l-arginine analogs indicates that the availability of l-arginine, the substrate for NOS, is important for the generation of NO (66). l-arginine is supplied by intracellular synthesis and/or uptake from the extracellular space. In the cytoplasm, l-arginine is converted from l-citrulline by argininosuccinate synthase or lyase. Arginase-2, the predominant isoform normally expressed in kidney, catalyzes hydrolysis of l-arginine to l-ornithine and urea and thus competes with NOS for l-arginine (Fig. 3). When elevated, arginase-2 can inhibit NOS activity and ex-

AA0 Na+ AA- 3Na+

Na+ AA+

EAAT3

B0 AT1

b0,+ AT

ASCT2

Apical

characterized by the urinary loss of l-arginine, l-ornithine, and l-lysine and by a poor intesinal absorption of these amino acids (60, 61). This leads to low plasma concentrations of these amino acids and to an impaired function of the urea cycle and hyperammonemia (62).

AA0 AA0 ATP AA+

AA0

NKA

TAT1

AA0

4F2hc LAT2

4F2hc y+LAT1

Basolateral

K+

AA0 AA-

AA0

ADP

Na+

4F2hc

LAT2

Figure 2. Epithelial AATs. The 4F2hc heavy chain can associate with a number of light chains to form different transporters, and its main role is the trafficking of the complex to the membrane. AA0, neutral amino acids; AA⫹, cationic amino acids; AA⫺, anionic amino acids. AMINO ACID TRANSPORTERS IN HYPERTENSION

5

Apical

Figure 3. Citrulline/NO cycle. Intracellular arginine, produced by citrulline metabolism or transported through CAT-1, is oxidized to citrulline and NO by NOS. Arginase catalyzes hydrolysis of l-arginine to l-ornithine and urea, thus competing with NOS for l-arginine. AG, arginase; ASS, synthase; ASL, arginosuccinate lyase.

L-citrulline

ASS

L-argininosuccinate

NO NOS

ASL

Urea L-ornithine

L-ARGININE

L-fumarate

CAT-1

AG

Basolateral

L-ARGININE

pression and thus induce endothelial NOS uncoupling, thereby reducing NO bioavailability and inhibiting the NO/cGMP pathway as well as increasing oxidative stress (67). System y⫹ is considered to be responsible for the majority of extracellular l-arginine, is widely distributed in a variety of tissues, and is encoded by the CAT (68). At least 4 CAT isoforms have been identified in mammals (CAT-1, CAT-2, CAT-2a, and CAT-3). CAT-1, CAT-2, and CAT-3 mRNA have been detected in the rat kidney. The expression of CAT-1 in the kidney is highest in the renal medulla, with little or no CAT-1 detectable in the renal cortex (68).

RENAL AATS AND HYPERTENSION Altered amino acid transport systems in the renal cortex The renal handling of L-DOPA and the regulation of amino acid transport systems in hypertension have been extensively studied in our laboratory. We reported that the tubular uptake of L-DOPA in Wistar Kyoto (WKY) rats and SHRs was a saturable process and was greater in SHRs than WKY rats at both 4 and 12 wk of age (69). While no significant differences were observed between Km values for the saturable component of L-DOPA uptake (393⫾68 vs. 110⫾30 ␮M for WKY vs. SHR, respectively), Vmax values were higher in SHRs than in WKY rats (542⫾40 vs. 786⫾68 pmol·mg protein⫺1·6 min⫺1 for WKY vs. SHR). Expression of LAT2 in renal cortex was higher in SHRs than in WKY rats. This increase was more marked at 4 than at 12 wk of age. It was suggested that overexpression of renal LAT2 in SHRs would result in enhanced production of renal dopamine, to compensate for the decreased dopaminemediated natriuresis generally observed in this genetic model of hypertension. Similarly, immortalized renal proximal tubular epithelial (PTE) cells from 4- to 8-wk-old prehypertensive SHRs were found to overexpress LAT2 (70). LAT1/4F2hc activity and expression has also been studied in these cells. The abundance of LAT1 and 4F2hc was greater in prehypertensive SHR than in WKY renal proximal tubular cells. However, this 6

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inversely correlated with the sodium-independent inward and outward transport of [14C]-l-leucine (70). In immortalized WKY PTE cells, L-DOPA uptake was almost exclusively through LAT2, whereas in immortalized SHR PTE cells, 50% of L-DOPA uptake occurred through LAT1, 25% through LAT2, and 25% through sodium-dependent mechanisms (70). These sodiumdependent mechanisms may involve ASCT2 and the neutral AAT B0AT1, which are the only transport systems that are capable of transporting amino acids with similar characteristics to substrates transported through system L. B0AT1 mRNA abundance in freshly isolated renal proximal tubules of SHRs was lower than that of normotensive WKY rats, at both 4 and 12 wk of age. Interestingly, the reduced B0AT1 mRNA levels in SHR kidney were associated with an increase in NHE3 expression, suggesting that increases in sodium tubular uptake might decrease B0AT1 gene transcription (71). Similarly, the abundance of ASCT2 transcript and protein in kidney cortices of SHRs was also lower than that in normotensive WKY rats. The saturable component of sodium-dependent l-alanine transport under Vmax conditions in SHR PTE cells was half of that in WKY PTE cells, with similar Km values (72). The rat SLC1A5 gene that codes for ASCT2 is located in chromosome 1 and has been previously associated with hypertension by several linkage analysis studies (73). In summary, in the renal cortex of 4-wk-old SHRs, LAT1 and LAT2 are overexpressed, while B0AT1 and ASCT2 are underexpressed. Therefore, these changes precede the onset of essential hypertension and may not occur simply as a consequence of the condition. However, it is likely that LAT2 overexpression may be an attempt to compensate for the decreased dopaminemediated natriuresis generally observed in SHRs and so may not be a cause of hypertension per se. On the other hand, the role of B0AT1 and ASCT2 underexpression in hypertension is not clear, and whether B0AT1 and ASCT2 overexpression could help prevent the onset of hypertension is not known. Recently, a knockout mouse model of B0AT3 was shown to have an abnormal excretion of several neutral amino acids and to develop high blood pressure under

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stress (74). However, in a cohort of 1004 Japanese individuals, a single polymorphism in SLC6A18 was not associated with hypertension or high blood pressure (75), and this lack of association was also confirmed in a Korean population (76). Another study has reported the association of a specific microsatellite marker in the SLC6A19 gene with essential hypertension although the underlying physiology is unclear (77). Altered amino acid transport systems in the renal medulla While our laboratory has focused mainly on the expression and activity of AATs in the renal cortex, others authors have explored how changes in l-arginine transport and NO metabolism in the renal medulla can contribute to the pathogenesis of hypertension. Wu et al. (78) have previously characterized l-arginine transport in inner medullary collecting duct cells from SD rat kidneys. The l-arginine transport system was saturable and inhibited by the cationic amino acids l-lysine, l-homoarginine, and l-ornithine, but was unaffected by the neutral amino acids l-leucine, l-serine, and l-glutamine. Both l-ornithine and l-lysine inhibited NOS enzymatic activity in a dose-dependent manner, supporting the important role of l-arginine transport for NO production. Chronic inhibition of l-arginine uptake in the renal medulla with a CAT-1 antisense oligonucleotide or by coadministration of other competing cationic amino acids led to a sustained reduction of NO and the development of hypertension (68). On the other hand, chronic intravenous infusion of the same dose of these cationic amino acids did not change NO in the medulla nor alter arterial pressure, suggesting that the in vivo production of NO and the regulation of MBF are partially dependent on the cellular uptake of l-arginine by a CAT-1. More recently, it was found that renal l-arginine transport is impaired in SHR (79). Renal [3H]-l-arginine transport in the SHR was lower than in SD rats. The kinetic analysis of renal [3H]-l-arginine uptake demonstrated a maximal velocity (Vmax) of 651 pmol·mg protein⫺1 ·min⫺1 and Km of 148 ␮M for SD rats. In the SHR, Vmax and Km of renal [3H]- l-arginine uptake were 456 pmol·mg protein⫺1 ·min⫺1 and 275 ␮M, respectively. Moreover, basal renal cortical and medullary NO content in the SHR was lower than in SD rats. Despite reductions in l-arginine transport, CAT-1 protein expression in the kidney in the SHR was significantly greater than in age-matched SD rats. The researchers suggested that the greater CAT-1 expression in the SHR kidney may reflect a compensatory increase in CAT-1 expression in response to low renal NO bioavailability in SHR. l-Arginine transport inhibitor, l-lysine, significantly reduced medullary NO content and tended to reduce medullary laser Doppler flux (MLDF) in SD rats but not in SHRs. Unlike in SHRs, superimposition of an infusion of l-arginine in l-lysine-pretreated SD rats increased MLDF and NO content to levels of control values (79). Therefore, although l-arginine transport AMINO ACID TRANSPORTERS IN HYPERTENSION

plays a critical role in modulating basal renal medullary perfusion and NO content in SD rats, it has little effect on renal medullary perfusion and NO concentration in the SHR. l-Lysine and subsequent l-arginine did not alter cortical laser Doppler flux in both SD rats and SHR, indicating that l-arginine transport may not have a significant role in regulating renal cortical perfusion in either rat strain. These results were consistent with previous findings by other authors. Arginine transport via the y⫹L system was shown to be lower in red blood cells from SHRs than in normotensive WKY rats (80). In addition, arginine transport was impaired in hypertensive and normotensive subjects genetically predisposed to hypertension, which suggested that impaired l-arginine transport may play a role in the development of this disease (81, 82). Whether decreased arginine transport in the adult SHR renal medulla may be a cause or a consequence of the hypertension is not known at this time. Interestingly, perinatal supplementation of l-arginine and antioxidants results in persistent reduction of systolic blood pressure and renal protection in the SHR (83). Although further research is required to evaluate the contribution of changes in the expression/activity of renal AATs to the development and progression of hypertension, it is apparent that the identification of genes responsible for this condition is an important prerequisite to prevent the expression of these disease markers related to lifestyle, especially for predisposed normotensives at risk.

MODULATION OF RENAL AATS IN HYPERTENSION Several factors have been shown to regulate the expression and/or activity of AATs. These factors include osmotic shock, growth factors, peptide hormones, and protein phosphorylation (84). In this review, we focus on the factors that potentially modulate AATs in the context of hypertension. Oxidative stress Several AATs have been reported to be regulated by reactive oxygen species. Oxidative stress has been found to stimulate the activity of NMDA receptors and the calcium-independent, carrier-mediated release of glutamate and [3H]-d-aspartate from cultured retina cells (85). On the other hand, one of most important features of system x⫺c is that its activity is highly inducible by various stimuli, including electrophilic agents like diethyl maleate, cystine deprivation in the culture medium, oxidized low density lipoprotein, and hydrogen peroxide (86). Recently, exposure of Caco-2 cells to the S-nitrosothiol type nitric oxide donor SNAP was found to increase sodium-dependent alanine uptake mediated by ASCT2 (87). Peroxynitrite stimulated sodium-dependent alanine transport and the NADPH oxidase inhibitor DPI, 7

and superoxide dismutase partially inhibited SNAPinduced sodium-dependent alanine transport, which suggested that NO-related radicals, as well as NO itself, might be responsible for stimulating ASCT2 (87). We attempted to explore the short- and long-term effects of L-NAME, SNAP, and carboxyl PTI on the inward transport of [14C]-l-alanine in monolayers of immortalized renal PTE cells from WKY rats and SHRs. No effects were observed with the tested concentrations and the treatment times (unpublished results), which may indicate that NO may not have a role in the regulation of ASCT2 in renal PTE cells. Previous studies in our laboratory have shown that apocynin, a NADPH oxidase inhibitor, significantly decreased Na⫹,K⫹-ATPase activity and Na⫹,K⫹-ATPase ␣1-subunit expression in OK cells (88). Furthermore, intracellular H2O2 can amplify the response downstream of ␣1-adrenoreceptor activation (89), ␣2-adrenoreceptor activation (90), and Cl⫺/HCO3⫺ exchanger activity (91) in immortalized SHR PTE cells. Therefore, we hypothesized that H2O2 or O2 could modulate ASCT2 activity. Lineweaver–Burk plots revealed the presence of high- and low-affinity states for the sodiumdependent [14C]-l-alanine uptake processes in both cell lines (72, 92). At low extracellular sodium concentrations, the sodium-dependent [14C]-l-alanine uptake in both WKY and SHR PTE cells is a high-affinity low-capacity process and increases in extracellular sodium reduced the affinity for the substrate but increased the capacity to take up [14C]-l-alanine. Inhibition of H2O2 production by apocynin during cell growth significantly reduced sodium Km and Vmax values of the low-affinity high-capacity component of sodium-dependent [14C]-l-alanine uptake in immortalized SHR PTE cells (92). Therefore, when H2O2 levels were reduced the sodium-dependent [14C]-l-alanine uptake by ASCT2 in SHR PTE cells functioned predominantly as a high-affinity low-capacity transporter. It was suggested that oxidative stress may have an effect on the conformations of ASCT2 in SHR PTE cells as they proceed through the transport cycle, which may result in differential sodium binding and unbinding. In fact, in immortalized SHR PTE cells, H2O2 has been shown to stimulate Cl⫺/HCO3⫺ exchanger activity via modification of thiol groups of intracellular and/or transmembrane proteins. The oxidized conformation of the exchanger enhanced the affinity for HCO3⫺ in immortalized SHR PTE cells but not in WKY PTE cells (93). Cysteine residues of proteins are especially susceptible to oxidative stress and, given the important role that disulfides play in protein structure and stability, alterations of reactive cysteine thiol groups may change protein function and activity.

high-salt diet (47). At 12 wk of age, high salt intake for 24 h increased urinary dopamine in SHRs, but not in WKY rats. Changes in urinary dopamine paralleled changes in the uptake of L-DOPA in isolated renal tubules from 4- and 12-wk-old WKY rats and SHRs with normal and high salt intake. At 12 wk of age, high salt intake was accompanied by decreases in LAT1 and LAT2 transcript abundance in WKY rats and SHRs. ASCT2 and B0AT1 expression was decreased in both 4and 12-wk-old WKY rats and in 4-wk-old SHRs with high salt intake. By contrast, high salt intake increased ASCT2 and B0AT1 expression in 12-wk-old SHRs (47). The results suggested that sodium-dependent transport systems ASCT2 and B0AT1 may be promoting the L-DOPA uptake, and this would be most prominent in the SHRs. Recently, collectrin has been implicated in the development of salt-sensitive hypertension. Collectrin mRNA isolated from renal cortex and medulla was up-regulated in 17-wk-old WKY rats and SHRs fed a high-salt diet (94). Up-regulation of collectrin by high salt intake was independent of aldosterone and linked to the trafficking of apical membrane proteins via the SNARE complex (94). Sodium-retaining hormones, such as aldosterone, have been shown to modulate AATs. Using differential display PCR to compare cDNA fragments generated from RNA of control and aldosterone-treated (40 and 60 min) epithelia, Spindler et al. (95) reported Xenopus leavis ASUR4 (homologue of human LAT1) as one of the early aldosterone up-regulated RNAs in A6 cells. Attempts were made to determine whether treatment with aldosterone had an effect on L-DOPA uptake and LAT2 expression in WKY and SHR PTE cells and in Madin-Darby canine kidney epithelial cells, but no differences were observed in the treatment time frame of the study (unpublished results). On the other hand, 8-wk-old normotensive Wistar rats chronically treated for 8 d with aldosterone had increased renal cortical LAT2 mRNA levels with no changes in LAT1, 4F2hc, and ASCT2 transcript levels (96). The effect of aldosterone on LAT2 mRNA levels was completely prevented by spironolactone, a mineralocorticoid receptor antagonist. At the protein level, aldosterone treatment did not significantly affect LAT1 and LAT2 expression, but markedly reduced the abundance of 4F2hc, although reduced levels were not reversed by spironolactone. The decrease in LAT2 functionality (related to the decrease of 4F2hc abundance) correlated well with the reduction in urinary dopamine (96). Taken together, these results suggested that the transcript abundance of AATs is age dependent and can be modulated by aldosterone.

Salt loading and salt-retaining hormones

Aging

Renal mRNA abundance of sodium-independent AATs LAT1 and LAT2, 4F2hc, and sodium-dependent AATs ASCT2 and B0AT1 were previously evaluated in 4- and 12-wk-old WKY rats and SHRs fed a normal salt or

Recently, ⬎500 genes were consistently found to be differentially expressed among human kidney samples from young, adult, and old subjects (97). The genes that showed altered expression in the older

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kidneys included those of 12 different solute carrier genes (transporters involved in tubular transport of electrolytes, ions, glucose, organic acids, and amino acids). Among these, 2 genes for AAT (SLC7A7 and SLC7A9) were shown to have decreased transcript expression with age (97). Recently, age-related changes in the renal cortical abundance of AATs potentially involved in renal tubular uptake of LDOPA were reported. LAT1 expression levels were down-regulated in aged WKY rats and SHRs when compared to young animals (98). Plasma levels of L-DOPA decreased in both WKY rats and SHRs with age (WKY: 13 wk, 4.07⫾0.31 pmol/ml, vs. 91 wk, 2.00⫾0.21 pmol/ml; SHR: 13 wk, 5.33⫾0.36 pmol/ ml, vs. 91 wk, 2.89⫾0.21 pmol/ml). Aging was accompanied by ASCT2 up-regulation in WKY rats and ASCT2 and 4F2hc/LAT2 up-regulation in SHRs. Moreover, SHRs had increased efficiency in the formation of renal dopamine, and this was accompanied an increase in the activity of the sympathetic and renin-angiotensin-aldosterone systems (98).

CONCLUSIONS Renal dysfunction underlies the development of hypertension in humans and experimental animals, and the handling of amino acids by the kidney is altered in this condition. To the best of our knowledge, we have summarized for the first time the renal transport systems that are altered during the onset, as well as the maintenance phases of essential hypertension. Our research efforts continue to focus on the molecular bases of renal reabsorption of amino acids and the pathophysiology of altered amino acid transport systems. Ongoing studies in our laboratory using a new technique to evaluate the role of LAT1, LAT2, ASCT2, and 4F2hc genes in Balb/c mice, which are normotensive but can become hypertensive with renal silencing of selected hypertensinogenic genes. This novel technique of organ-restricted silencing of gene expression using gene-specific siRNA infused into the organ (e.g., kidney) offers the advantage of restricted silencing of the gene of interest to relevant tissues only and precludes unforeseen compensatory mechanisms that may develop in systemic-knockout mouse models. We are also currently investigating other factors, such as obesity, that may influence AAT activity/expression. The identification of factors that influence renal AAT function would potentially be beneficial to prevent the development of hypertension. Efforts should also be made to determine whether resurrecting renal arginine transport may help restore renal NO content and improve renal endothelial function. In addition, there are other questions that remain unanswered and that deserve future attention, such as whether mutations are responsible for the low transporter activities of CAT-1 and whether changes in renal amino acid transport AMINO ACID TRANSPORTERS IN HYPERTENSION

expression/function are observed in human subjects with hypertension. Phenotype-driven rat experimental models, such as SHRs, are the most abundant for hypertension research. However, new technologies may be required in order to extend the findings to humans. Once the role of AATs in hypertension has been thoroughly investigated, other challenges in targeting renal amino acid for diagnostic or therapeutic purposes may remain. Although genetic linkage and candidate gene association studies have implicated various loci and genes in hypertension, consistent associations have been difficult to demonstrate mainly because of the ethnic divergence of gene polymorphisms. The advantage of DNA testing for polymorphisms and determining the genotype of a patient lies in the fact that it may predict response to a certain class of antihypertensive agent and thus optimize therapy in individual patients. Additionally, targeting altered AATs by gene therapy could potentially be a viable strategy for the long-term control of hypertension. Some of the challenges that need to be resolved include ideal gene transfer vector, precise delivery of genes into the required site, efficient transfer of genes into the cells of the target, and prompt assessment of gene expression over time. However, although studies in animals indicate that gene therapy may be feasible in treating hypertension, this may not be feasible in the near future for humans. This work was supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Ciência e Inovação (POCI), Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Comunitário de Apoio (PIC/IC/83204/2007). The authors declare no conflicts of interest.

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Received for publication February 18, 2013. Accepted for publication April 8, 2013.

PINTO ET AL.