BASIC RESEARCH www.jasn.org Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids Clara Vilches ,1 Emilia Boiadjieva-Knöpfel,2,3,4 Susanna Bodoy ,5,6 Simone Camargo ,2,3,4 Miguel López de Heredia ,1,7 Esther Prat ,1,7,8 Aida Ormazabal ,7,9 Rafael Artuch ,7,9 Antonio Zorzano ,5,6,10 François Verrey ,2,3,4 Virginia Nunes ,1,7,8 and Manuel Palacín 5,6,7 1Molecular Genetics Laboratory, Genes Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), L’Hospitalet de Llobregat, Spain; 2Department of Physiology, 3Zurich Center for Integrative Human Physiology (ZIHP), and 4Swiss National Centre of Competence in Research (NCCR), Kidney Control of Homeostasis (Kidney.CH), University of Zurich, Zurich, Switzerland; 5Department of Biochemistry and Molecular Medicine, Biology Faculty, University of Barcelona, Barcelona, Spain; 6Molecular Medicine Unit, Amino acid transporters and disease group, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain; 7Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) – U730, U731, U703, and 10Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) – CB07/08/0017, Instituto de Salud Carlos III (ISCIII), Madrid, Spain; 8Genetics Section, Physiological Sciences Department, Health Sciences and Medicine Faculty, University of Barcelona, Barcelona, Spain; and 9Clinical Biochemistry Department, Institut de Recerca Sant Joan de Déu, Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain ABSTRACT Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which me- diates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooper- ation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo. Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double- knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc. Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA trans- porters may explain the lack of basolateral neutral aminoacidurias in humans. J Am Soc Nephrol 29: 1624–1635, 2018. doi: https://doi.org/10.1681/ASN.2017111205 Received November 21, 2017. Accepted February 24, 2018. Biomedicine-Barcelona, Baldiri i Reixac 10, 08028 Barcelona, Spain, or Dr. Virginia Nunes, Institut d’Investigació Biomèdica de C.V., E.B.-K., and S.B. contributed equally to this work. Bellvitge (IDIBELL), Gran Via de L’Hospitalet 199, L’Hospitalet de Llobregat, 08908 Barcelona, Spain, E-mail: manuel.palacin@ F.V., V.N., and M.P. shared senior authorship. irbbarcelona.org or [email protected] Published online ahead of print. Publication date available at www.jasn.org. Copyright © 2018 by the American Society of Nephrology Correspondence: Dr. Manuel Palacín, Institute for Research in 1624 ISSN : 1046-6673/2906-1624 JAmSocNephrol29: 1624–1635, 2018 www.jasn.org BASIC RESEARCH Renal reabsorption accounts for .98% of recovery of most Significance Statement circulating amino acids (AAs) filtered in the glomerulus. To this end, AAs are actively transported across epithelial cells of Renal amino acid reabsorption is crucial for the maintenance of the renal proximal tubule by AA transporters located in their whole body homeostasis and its impairment leads to several dis- apical and basolateral membranes1 (Figure 1A). Primary in- eases such as cystinuria and Hartnup disorder. Whereas these and otherwell describedaminoacidurias are causedbydefectsof luminal herited aminoacidurias caused by loss-of-function mutations transport proteins, only one aminoaciduria, specifically of cationic demonstrate the role of some transporters in renal reabsorp- amino acids, is associated with the dysfunction of a basolateral tion.2 Thus, mutations in the apical transporters rBAT/b0,+AT transporter. This work demonstrates in vivo the functional co- 3,4 5,6 operation of two basolateral neutral amino acid transporters, LAT2 (SLC3A1/SLC7A9), B0AT1 (SLC6A19), EAAC1 + (SLC1A1),7 and PAT2 (SLC36A2) alone or in combination and TAT1, and shows that another basolateral transporter, y LAT1, can largely compensate for their defect. These findings reveal 8 with mutations in IMINO (SLC6A20) cause cystinuria, synergistic and compensatory reabsorption mechanisms in renal Hartnup disorder, dicarboxylic aminoaciduria, and iminogly- epithelial cells that can explain why no neutral aminoaciduria due cinuria, respectively. Knockout mouse models of these trans- to the defect of basolateral transporters has been identified in porters mimic these human aminoacidurias.9–12 humans. The molecular bases of renal reabsorption of AAs at the baso- lateral membrane are less well understood. The study of the only METHODS known human aminoaciduria involving a basolateral transporter + demonstrated the role of y LAT1/CD98hc (SLC7A7/SLC3A2)in A full, detailed description of all materials and methods used is lysinuric protein intolerance and in renal reabsorption of cationic provided in the Supplemental Material. AA.13,14 Ablation of y+LAT1 also resulted in cationic aminoacid- 15 uria and large neonatal lethality in mice. Mouse Model Generation, Genotyping, and For neutral AAs, three knockout mouse models of basolateral Experimental Diets transporters have been reported, two of which showed a mild Single loss-of-function mouse models for LAT2 (null knockout)24–26 phenotype in AA renal reabsorption, whereas the third (LAT4 and TAT1 (premature STOP codon at position Y88)17 were crossed 16 [Slc43a2] knockout) was postnatally lethal. Ablation of the aro- to obtain double heterozygous mice and backcrossed to get the matic AA uniporter TAT1 (Slc16a10) caused, next to a substantial F2 generation, including dKO LAT2-TAT1 (dKO) mice. Geno- aromatic aminoaciduria, also a moderate aminoaciduria of other type was confirmed by PCR and/or Sanger sequencing. For 17 neutral AAs that became exacerbated under a protein-rich diet. exacerbation of renal phenotype, a protein-rich diet was used It is interesting to note that the plasma concentration of aromatic (40% casein). As control, mice were fed with a 20% casein diet. AAs was strongly increased despite their urinary loss, presumably C57BL/6J mice were maintained in a 12-hour light/dark cycle, due to the absence of TAT1 from hepatocytes that normally func- with free access to food and water. Experimental diets were 17 tion as a metabolic sink for aromatic AAs. The knockout of maintained for 8 days, with free access to water. Males and LAT2, the catalytic subunit of LAT2/CD98hc (Slc7a8/Slc3a2)het- females were used for initial characterization of the dKO mouse erodimer, showed a very mild aminoaciduria.18 Redundancy and model; only male mice were included in renal function studies. compensatory mechanisms most probably underlie these mild phenotypes of hyperexcretion of AAs in urine. Mouse Sample Collection and Analyses TAT1 and LAT2/CD98hc have been shown to functionally co- Mice were individually housed in metabolic cages for 4 days during 19 operate in a cellular model. TAT1 is a uniporter that mediates which experimental diet was maintained. Twenty-four-hour urine 20,21 downhill transport of L-aromatic AAs (Tyr, Phe, and Trp) and samples, blood plasma, and organs of interest were harvested. AAs LAT2/CD98hc is an obligatory exchanger of any L-neutral AA be- and creatinine were determined in urine and plasma. Kidney total 22,23 side proline. ThecoexpressionofTAT1wasshowntoenable RNA was analyzed by RT-qPCR by using UPL probes (Roche Life- fl glutamine to ef ux via LAT2/CD98hc in exchange with aromatic Science, Switzerland) in a microfluidic chip (Fluidigm, CA). LAT2, fl 19 AAs ef uxed via TAT1 in Xenopus oocytes. TAT1 and LAT2 are TAT1, and y+LAT1 proteins were detected from total membranes expressed in the basolateral membrane of the same epithelial cells in extracted from kidneys as detailed in the Supplemental Material. the kidney proximal tubule.19 Thus, these two transporters might cooperate in renal reabsorption using common substrates (Figure 1A). To study their functional relationship in vivo,adouble-knock- RESULTS out mouse model (dKO LAT2-TAT1) has been generated. Here, we show that dKO LAT2-TAT1 mice presented a synergistic defect of General Features of the Double-Knockout