Plasma Urate Level Is Directly Regulated by a Voltage-driven Urate Efflux Transporter URATv1 (SLC2A9) in Humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (Km: 365 ± 42 μm). The transport was Na+-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; Remunurate is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1). Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (Km: 365 ± 42 μm). The transport was Na+-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; Remunurate is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1). Urate (uric acid), an end product of purine metabolism in humans because of the genetic silencing of hepatic uricase, is now recognized as a natural antioxidant that has neuroprotective properties (1. Kutzing M. K. Firestein B. L. J. Pharmacol. Exp. Ther. 2008; 324: 1-7Crossref PubMed Scopus (252) Google Scholar). Despite its beneficial role, elevation of the serum urate level is correlated with gout, hypertension, and cardiovascular and renal diseases (1. Kutzing M. K. Firestein B. L. J. Pharmacol. Exp. Ther. 2008; 324: 1-7Crossref PubMed Scopus (252) Google Scholar, 2. Becker M. A. Jolly M. Rheum. Dis. Clin. N. Am. 2006; 32: 275-293Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The kidney plays a dominant role in maintaining plasma urate levels through the excretion process; it eliminates ∼70% of the daily urate production (3. Sica D. A. Schoolwerth A. C. The Kidney. 6th Ed. WB Saunders, Philadelphia2000: 680-700Google Scholar). Therefore, it is important to understand the mechanism of renal urate handling because underexcretion of urate has been demonstrated in the majority of hyperuricemia patients (4. Mount D. B. Kwon C. Y. Zandi-Nejad K. Rheum. Dis. Clin. N. Am. 2006; 32: 313-331Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Since urate is a weak acid at physiological pH (pKa, 5. 75), it hardly permeates the plasma membrane of cells in the absence of transport proteins (3. Sica D. A. Schoolwerth A. C. The Kidney. 6th Ed. WB Saunders, Philadelphia2000: 680-700Google Scholar). In 2002, we identified a long hypothesized urate-anion exchanger, URAT1, 2The abbreviations used are: URAT1, urate transporter 1; URATv1, voltage-driven urate transporter 1; SLC, solute carrier; GLUT9, glucose transporter 9; PZA, pyrazinoate; DIDS, 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid; MES, 4-morpholineethanesulfonic acid. 2The abbreviations used are: URAT1, urate transporter 1; URATv1, voltage-driven urate transporter 1; SLC, solute carrier; GLUT9, glucose transporter 9; PZA, pyrazinoate; DIDS, 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid; MES, 4-morpholineethanesulfonic acid. encoded by SLC22A12, that localized on the apical side of the renal proximal tubule (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). Despite several potential candidate proteins for urate transport such as UAT (uric acid transporter), OAT1 (organic anionic transporter 1), OAT3, OAT4, OATv1/NPT1 (sodium phosphate transporter 1), MRP4 (multidrug resistance-associated protein), and OAT10 (6. Rafey M. A. Lipkowitz M. S. Leal-Pinto E. Abramson R. G. Curr. Opin. Nephrol. Hypertens. 2003; 12: 511-516Crossref PubMed Scopus (85) Google Scholar, 7. Hediger M. A. Johnson R. J. Miyazaki H. Endou H. Physiology (Bethesda). 2004; 20: 125-133Google Scholar, 8. Anzai N. Enomoto A. Endou H. Curr. Rheumatol. Rep. 2005; 7: 227-234Crossref PubMed Scopus (54) Google Scholar, 9. Eraly S. A. Vallon V. Rieg T. Gangoiti J. A. Wikoff W. R. Siuzdak G. Barshop B. A. Nigam S. K. Physiol. Genomics. 2008; 33: 180-192Crossref PubMed Scopus (182) Google Scholar, 10. Bahn A. Hagos Y. Reuter S. Balen D. Brzica H. Krick W. Burckhardt B. C. Sabolic I. Burckhardt G. J. Biol. Chem. 2008; 283: 16332-16341Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), URAT1 is the sole transporter whose physiological role in renal urate reabsorption is established, based on the fact that loss-of-function mutations in URAT1 cause renal hypouricemia (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). However, it is not known how urate taken up via URAT1 exits from the tubular cell (11. Anzai N. Kanai Y. Endou H. Curr. Opin. Rheumatol. 2007; 19: 151-157Crossref PubMed Scopus (161) Google Scholar). Moreover, there are patients with renal hypouricemia who had no mutation in SLC22A12, suggesting the existence of a non-URAT1-mediated urate reabsorption system (12. Ichida K. Hosoyamada M. Hisatome I. Enomoto A. Hikita M. Endou H. Hosoya T. J. Am. Soc. Nephrol. 2004; 15: 164-173Crossref PubMed Scopus (313) Google Scholar, 13. Wakida N. Tuyen D. G. Adachi M. Miyoshi T. Nonoguchi H. Oka T. Ueda O. Tazawa M. Kurihara S. Yoneta Y. Shimada H. Oda T. Kikuchi Y. Matsuo H. Hosoyamada M. Endou H. Otagiri M. Tomita K. Kitamura K. J. Clin. Endocrinol. Metab. 2005; 90: 2169-2174Crossref PubMed Scopus (52) Google Scholar). Here we report a previously unknown urate transporter on the basolateral side of the renal proximal tubule, which is likely to act in tandem with URAT1 for urate reabsorption in its physiological role in vivo in humans. Immunohistochemistry in Xenopus Oocytes—cRNA synthesis was performed as described elsewhere (14. Islam R. Anzai N. Ahmed N. Ellapan B. Jin C. J. Srivastava S. Miura D. Fukutomi T. Kanai Y. Endou H. J. Pharmacol. Sci. 2008; 106: 525-528Crossref PubMed Scopus (21) Google Scholar). Xenopus laevis oocytes injected with cRNAs were fixed with paraformaldehyde and incubated with the anti-GLUT9 antibody (Alpha Diagnostics) (1: 500) followed by Alexa Fluor 546-labeled goat anti-rabbit immunoglobulin (IgG) (Wako; diluted 1: 200), as described previously (15. Yokoyama H. Anzai N. Ljubojevic M. Ohtsu N. Sakata T. Miyazaki H. Nonoguchi H. Islam R. Onozato M. Tojo A. Tomita K. Kanai Y. Igarashi T. Sabolic I. Endou H. Cell Physiol. Biochem. 2008; 21: 269-278Crossref PubMed Scopus (27) Google Scholar). The sections were observed under a confocal laser scanning microscope (Fluoview FV500, Olympus). Functional Characterization of GLUT9—GLUT9 isoform 1 and 2 cDNAs were purchased from OriGene Technologies (isoform 2) and Open Biosystems (isoform 1). In vitro transcription and injection of capped cRNA into oocytes were performed as described previously (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar, 16. Jutabha P. Kanai Y. Hosoyamada M. Chairoungdua A. Kim D. K. Iribe Y. Babu E. Kim J. Y. Anzai N. Chatsudthipong V. Endou H. J. Biol. Chem. 2003; 278: 27930-27938Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Oocytes were maintained in Barth's buffer for 2–3 d at 18 °C before use. The ND96 buffer contained (in mm) 96 NaCl, 2 KCl, 1 MgCl2, 1. 8 CaCl2, and 5 HEPES buffer (pH 7. 4). In the 0 Cl– bath, Cl– was replaced with an equimolar amount of gluconate. Kinetic parameter for the uptake of urate was estimated from the following equation: v = Vmax × S/ (Km + S), where v is the rate of substrate uptake (pmol/h/oocyte), S is the substrate concentration in the medium (μm), Km is the Michaelis-Menten constant (μm), and Vmax is the maximum uptake rate (pmol/h/oocyte). These kinetic parameters were determined by the Eadie-Hofstee plot. The trans-stimulation experiments were done as described previously (17. Anzai N. Jutabha P. Enomoto A. Yokoyama H. Nonoguchi H. Hirata T. Shiraya K. He X. Cha S. H. Takeda M. Miyazaki H. Sakata T. Tomita K. Igarashi T. Kanai Y. Endou H. J. Pharmacol. Exp. Ther. 2005; 315: 534-544Crossref PubMed Scopus (82) Google Scholar). The experiments were performed using three batches of oocytes, and results from the representative experiments are expressed as mean ± S. E. Statistical significance was determined by Student's t test. Mutation Analysis and Construction of Mutant cDNA—For the GLUT9 sequence determination in renal hypouricemia patients and normal control subjects, we used the primers described by S. Li with slight modification (18. Li S. Maschio A. Busonero F. Usala G. Mulas A. Lai S. Dei M. Orrù M. Albai G. Bandinelli S. Schlessinger D. Lakatta E. Scuteri A. Najjar S. S. Guralnik J. Naitza S. Crisponi L. Cao A. Abecasis G. Ferrucci L. Uda M. Chen W. M. Nagaraja R. PLoS Genet. 2007; 3: e194Crossref PubMed Scopus (230) Google Scholar). Institutional approval was obtained at each participating site. High molecular weight genomic DNA was extracted from peripheral whole blood cells and was amplified by PCR. The PCR products were sequenced in both directions using a 3130xl genetic analyzer (Applied Biosystems). To generate a GLUT9 mutant (P412R), we performed site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions (19. Sakata T. Anzai N. Shin H. J. Noshiro R. Hirata T. Yokoyama H. Kanai Y. Endou H. Biochem. Biophys. Res. Commun. 2004; 313: 789-793Crossref PubMed Scopus (93) Google Scholar). The mutagenic oligonucleotide primers for generation of P412R mutant were 5′-CACGCCCCCTGGGTCCGCTACCTGAGTATCGTG-3′ (forward) and 5′-CACGATACTCAGGTAGCGGACCCAGGGGGCGTG-3′ (reverse). Proper construction of the mutated cDNA was confirmed by complete sequencing. To identify novel renal urate transporters, we performed a homology search (Blastp) against the Swiss-Prot protein data base in the National Center for Biotechnology Information (NCBI) using the human and mouse sequences for URAT1/Urat1 (SLC22A12/Slc22a12) (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar, 20. Mori K. Ogawa Y. Ebihara K. Aoki T. Tamura N. Sugawara A. Kuwahara T. Ozaki S. Mukoyama M. Tashiro K. Tanaka I. Na-kao K. FEBS Lett. 1997; 417: 371-374Crossref PubMed Scopus (71) Google Scholar) and human OAT4 (SLC22A11) (21. Cha S. H. Sekine T. Kusuhara H. Yu E. Kim J. Y. Kim D. K. Sugiyama Y. Kanai Y. Endou H. J. Biol. Chem. 2000; 275: 4507-4512Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Surprisingly, we found that several members of the facilitated glucose transporter (SLC2) family (GLUT6, -9, -10, -12, and -14) have remote similarities to SLC22A11. Among these molecules, we decided to characterize one of the extended (class II) SLC2 family members named GLUT9 (which is encoded by SLC2A9) because it localizes mainly in the kidney and the liver (22. Uldry M. Thorens B. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 480-489Crossref PubMed Scopus (385) Google Scholar). Human GLUT9 was originally identified as a gene of unknown function (23. Phay J. E. Hussain H. B. Moley J. F. Genomics. 2000; 66: 217-220Crossref PubMed Scopus (173) Google Scholar). Although glucose transporter activity of GLUT9 was demonstrated, it does not seem as efficient as the classical (class I) glucose transporter GLUT4 (24. Doege H. Bocianski A. Joost H. -G. Schurmann A. Biochem. J. 2000; 350: 771-776Crossref PubMed Scopus (138) Google Scholar, 25. Augustin R. Carayannopoulos M. O. Dowd L. O. Phay J. E. Moley J. F. Moley K. H. J. Biol. Chem. 2004; 279: 16229-16236Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Human GLUT9 has two splice variants: isoform 1 (NM₀20041) and isoform 2 (NM₀01001290) (supplemental Fig. S1A). The difference between these two isoforms lies in the presence of the first exon only in isoform 1, which results in differential targeting in polarized Madin-Darby canine kidney cells (25. Augustin R. Carayannopoulos M. O. Dowd L. O. Phay J. E. Moley J. F. Moley K. H. J. Biol. Chem. 2004; 279: 16229-16236Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). First, we examined the membrane expression and urate transport activities of both isoforms of GLUT9 using the Xenopus oocyte expression system. Both isoforms of GLUT9 were expressed on the plasma membrane when GLUT9 cRNAs were injected into oocytes (supplemental Fig. S2A), and both isoforms had equivalent urate transport activity (supplemental Fig. S2B). We used isoform 2 for further characterization. The uptake rate of 14Curate in oocytes expressing GLUT9 was 9-fold higher than that in control oocytes, whereas much lower uptake rates of 14Cglucose and 14Cfructose were observed (supplemental Fig. S2C). GLUT9 did not show any significant uptake of representative organic anionic substrates such as para-aminohipurate, estrone sulfate, or salicylate, nor of substrates known as of classical renal urate transport such as or (supplemental Fig. S2C). GLUT9 had a substrate specificity than URAT1. we examined the urate transport properties of Xenopus oocytes expressing GLUT9 transport of 14Curate The uptake of urate saturable and followed the Michaelis-Menten The Eadie-Hofstee a Km of 365 ± 42 and a Vmax of ± ± S. E. of for that GLUT9 has a high for urate to URAT1 of extracellular did not urate transport by GLUT9, that it did not a urate GLUT9 activity was to membrane potential because the elevation of (which the plasma membrane of a Xenopus facilitated urate uptake sensitivity the efflux of urate from the tubular cells because of a negative the cell. We the of an Cl– to on urate a Cl– by complete of Cl– did not the urate uptake via GLUT9, that GLUT9 does not have the mechanism for Cl– observed in URAT1 In URAT1, a of urate transport activity on extracellular pH was observed for GLUT9 To further the substrate of GLUT9, an was The of at 1 for at on 14Curate uptake was examined 1). GLUT9 properties from those of URAT1 (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). Although the urate transport via GLUT9 was by such as glucose and such as and or such as and did not urate uptake via These results are with the that urate to be the only substrate for GLUT9 (supplemental Fig. of 14Curate uptake by The uptake in oocytes was determined at 1 in the absence or presence of to the extracellular medium (pH 7. 4). The were expressed as of uptake under control are mean ± S. E. with = with the uptake in of ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Open in a We the of (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar, N. J. PubMed Google Scholar). and urate uptake pyrazinoate the of used as an and known as an with URAT1 (5. Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S. H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimokata K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar), did not any on urate uptake via an urate These results demonstrated that it to renal urate handling from that of URAT1, that GLUT9 be a potential for novel To the transport of GLUT9, we performed trans-stimulation experiments by organic into oocytes to 14Curate urate mm) 14Curate uptake via GLUT9 (supplemental Fig. from the of on urate uptake by PZA, and salicylate, of these had no (supplemental Fig. with that GLUT9 act as an efflux transporter under a physiological oocytes with 14Curate a efflux of when incubated in the uptake its basolateral membrane in proximal tubular cells (25. Augustin R. Carayannopoulos M. O. Dowd L. O. Phay J. E. Moley J. F. Moley K. H. J. Biol. Chem. 2004; 279: 16229-16236Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), we propose that GLUT9 is for urate from the cell to the as a in reabsorption following the mediated by URAT1 the efflux of from the oocytes with 14Curate was in the absence and presence of extracellular and the efflux was by the extracellular urate not by glucose or (supplemental Fig. Therefore, urate transport via GLUT9 not be by the normal serum levels of these renal hypouricemia renal in is a by renal urate and by an of membrane transport for urate in the renal proximal tubule O. Genet. Metab. 2006; PubMed Scopus Google Scholar). there are renal hypouricemia patients who have no mutation in SLC22A12 (12. Ichida K. Hosoyamada M. Hisatome I. Enomoto A. Hikita M. Endou H. Hosoya T. J. Am. Soc. Nephrol. 2004; 15: 164-173Crossref PubMed Scopus (313) Google Scholar, 13. Wakida N. Tuyen D. G. Adachi M. Miyoshi T. Nonoguchi H. Oka T. Ueda O. Tazawa M. Kurihara S. Yoneta Y. Shimada H. Oda T. Kikuchi Y. Matsuo H. Hosoyamada M. Endou H. Otagiri M. Tomita K. Kitamura K. J. Clin. Endocrinol. Metab. 2005; 90: 2169-2174Crossref PubMed Scopus (52) Google Scholar). GLUT9 in the urate reabsorption in tandem to URAT1, it is that such patients in the of these patients had a plasma urate level of normal is to the found in renal hypouricemia patients with a URAT1 mutation such as Analysis of the from the genomic DNA of the patient the presence of a to at exon of (supplemental and to was located in the extracellular side (supplemental Fig. and was in both and GLUT9 in SLC2 family members H. -G. Thorens B. Biol. PubMed Scopus Google Scholar). P412R mutation was not identified in control To the function of mutant GLUT9, we urate transport activity injection of or mutated GLUT9 cRNA into oocytes and found that reduced urate transport activity level of both and mutant GLUT9 was in the plasma membrane by In a sugar transport facilitator family protein GLUT9 was found to act as a voltage-driven urate is for urate efflux transporter from the cell. complete a model of urate reabsorption in the renal tubular where urate in the is taken up via URAT1 and urate exits from the cell to the via GLUT9 Based on model we that function of both URAT1 and GLUT9 is for normal urate reabsorption in the renal proximal tubule both act on the in was supported in vivo by the existence of mutations with reduced function in in a patient with renal hypouricemia who had no mutations in mutation a with a we that it the of GLUT9 with its substrate we were in the of of GLUT9, a genetic have been that to the plasma urate Li (18. Li S. Maschio A. Busonero F. Usala G. Mulas A. Lai S. Dei M. Orrù M. Albai G. Bandinelli S. Schlessinger D. Lakatta E. Scuteri A. Najjar S. S. Guralnik J. Naitza S. Crisponi L. Cao A. Abecasis G. Ferrucci L. Uda M. Chen W. M. Nagaraja R. PLoS Genet. 2007; 3: e194Crossref PubMed Scopus (230) Google Scholar) the of serum urate levels with genetic in the In results to two whole that demonstrated that the serum urate levels were correlated with in or to A. D. H. H. S. G. K. N. F. B. A. D. H. T. T. Genet. 2008; PubMed Scopus Google Scholar, V. I. J. S. A. I. O. J. J. F. A. X. B. N. B. J. S. L. M. L. T. S. H. P. A. M. S. H. P. H. Genet. 2008; PubMed Scopus Google Scholar). proteins encoded by as urate in the mainly to urate from the tubular and urate transport activity the plasma urate Therefore, we propose that the proteins encoded by be URATv1 (voltage-driven urate transporter 1), of We the patients in We A. M. and N. Ohtsu for Xenopus oocyte R. K. and A. for H. Matsuo for patient and A. Enomoto for of the with