sglt2 inhibitor lowers serum uric acid through alteration.pdf

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and possibly to modulate uric acid (UA) transportin healthy subjects [11]. However, the exactmechanism of the uricosuric effect of phloridzinremains unclear.

Elevated serum uric acid levels are associated

with gout attacks [12,13]. In addition, recentevidence suggests a signicant association be-tween hyperuricemia and chronic kidney disease[14,15], metabolic syndrome [16,17], hypertension[18,19] or cardiovascular events [20,21]. Theserum uric acid levels depend on a balance be-tween the production and excretion of uric acid,in which approximately 70% of the uric acid isexcreted into the urine and the remainder isthought to be excreted into the intestinal tract.The renal transport system plays an importantrole in the regulation of the SUA level and in-

volves reabsorption and secretion. Consequently,~90% of glomerularly ltered uric acid isreabsorbed by the epithelial cells of the proximaltubules [22,23]. Recently, transporters involved inthe renal handling of uric acid have been identi-ed. Uric acid is reabsorbed by the proximaltubule cells mainly via the uric acid transporter 1(URAT1,SLC22A12) [24,25], possibly via organicanion transporter 4 (OAT4, SLC22A11) [26,27]and OAT10 (SLC22A13) [27], and is transportedto the blood via GLUT9 isoform 1 (SLC2A9a)[25,2831]. Furthermore, uricosuric agents (suchas benzbromarone and probenecid), angiotensin IIreceptor blockers (such as losartan) and salicylicacid inhibit URAT1, and this explains the SUA-lowering effects of these drugs [24,32]. To date, theeffects of SGLT inhibitors on transporters involvedin the renal handling of uric acid are unknown.

Hyperuricemia is closely linked to diabetes,since insulin resistance is correlated with SUAlevels but inversely correlated with the renal clear-ance of curic acid (CLUA) [33,34]. Insulin has beensuggested to increase uric acid reabsorption in the

proximal tubule [34]. On the other hand, when thedisease progresses to the stage of glycosuria, theserum uric acid level begins to decrease [3538].These mechanisms have been proposed as aneffect of glucose on uric acid handling in theproximal tubule [35,37]. This phenomenon is simi-lar to the SUA-lowering effect of SGLT2 inhibitors.

The present study describes the SUA-loweringeffect of luseogliozin in clinical studies to assessthe safety, pharmacokinetics and glucose-excreting

effect of luseogliozin in healthy subjects [39]. Inaddition, in vitro experiments were performedusing Xenopus oocytes and cultured cells expressinguric acid transporters to elucidate the mechanism.

Materials and Methods

Subjects and study design

Fifty-seven and 24 healthy Japanese men partici-pated in a single dose study and a multiple dosestudy of luseogliozin, respectively (Table 1).The subjects were between the ages of 20 and 39years and were in good health as assessed byscreening examinations, a complete medicalhistory and a physical examination. Subjects were

excluded from the studies if they had anyclinically signicant disease or had experienced asignicant body weight change (3 kg) within 4weeks of the rst administration or the use ofany drugs within the rst week of the rst admin-istration. All subjects provided written informedconsent prior to their participation in the studies.For the single dose study, 57 eligible individualswere randomly assigned to the following treatmentgroups: placebo, 1, 3, 5, 9, 15 or 25 mg luseogliozin.After fasting for at least 10 h, luseogliozin wasadministered orally. For the multiple dose study,24 eligible individuals were randomly assignedto the following treatment groups: placebo, 5 or10 mg. Luseogliozin was administered orallybefore breakfast once daily for 7 days. Blood andurine samples were collected at the predeterminedtime points. The studies complied with the Helsinki

Table 1. Demographic and baseline characteristics

Clinical characteristic Single dose study Multiple dose study

Subjects (males) 57 24

Age (years) 26.5 5.5 28.9 5.9Body weight (kg) 61.8 5.9 62.4 6.8HbA1c (%) 5.1 0.2 5.1 0.2HbA1c (mmol/mol) 32 3 32 3FPG (mg/dl) 91.1 4.8 89.3 5.7UEGL(g/day) 0.147 0.206 0.153 0.169SUA (mg/dl) 5.7 0.8 5.5 0.9UEUA(mg/day) 556 90 589 91CLUA(ml/min) 6.88 1.29 7.68 2.20

FPG, fasting plasma glucose;UEGL, urinary excretion rate of glucose;SUA, serum uric acid; UEUA, urinary excretion rate of uric acid; CLUA,

renal clearance of uric acid. Data are mean SD.

392 Y. CHINOET AL.

2014 The Authors.Biopharmaceutics & Drug Disposition.Published by John Wiley & Sons Ltd.

Biopharm. Drug Dispos.35: 391404 (2014)DOI: 10.1002/bdd


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Declaration, the standards of the Japanese Phar-maceutical Affairs Law, and the Good ClinicalPractice guidelines. The study protocols wereapproved by an Institutional Review Board(Kyushu Clinical Pharmacology Research Clinic,

Fukuoka, Japan). The studies were registeredwith the Japan Pharmaceutical Information Cen-ter (JapicCTI-132353, JapicCTI-132354) [39].

Measurements

Blood was centrifuged (4 C, 3000 rpm, 15 min) im-mediately after collection to obtain plasma samples.The concentrations of glucose and uric acid in theplasma and urine were measured using anautomatic analyser (TBA-120FR, Toshiba MedicalSystems Corp. or Glucoroder-NX, A&T Corp.).

Concentrations of luseogli

ozin in the plasma andurine were determined using HPLC tandem massspectrometry (API4000; Applied Biosystems/MDSSciex, Foster City, CA, USA) after pre-treatmentwith solid phase extraction (OASIS HLB, WatersCorp., Milford, MA, USA). The lower limit of quan-tication was 0.05 ng/ml for plasma and 0.5 ng/mlfor urine, respectively. The CLUA were calculatedfrom the plasma concentration and urinary excre-tion data. TheAUC024hof the plasma luseogliozinconcentrations was calculated using a standardnoncompartmental method.

Transporter inhibition experiments in oocytes

The preparation of Xenopus laevis oocytes, thein vitro synthesis of cRNA (GLUT9 isoform 1,OAT10 or SMCT1) and the uptake experimentsusing [14C]UA (20 M) (except for SMCT1: [3H]nicotinic acid, 15M) were conducted as describedpreviously [4043]. For the inhibition experiment,uptake was initiated by replacement with a trans-port buffer (96 mMNaCl, 2 mMKCl, 1 mMMgCl2,1.8 mM CaCl2 an d5mM HEPES, pH 7.4) containing

[

14

C]UA or [

3

H]nicotinic acid and luseogli

ozin(Taisho Pharmaceutical, Saitama, Japan); for eachcondition, 810 oocytes prepared from a singlebatch, were incubated for 60 min at 25 C. The up-take reaction was terminated by washing the oocyteswith ice-cold transport buffer, and the oocytes werethen solubilized in 5% SDS for the quantication ofradioactivity. The uptake rate (l/min/oocyte)was calculated by dividing the uptake amount bythe initial concentration of the substrate in the

transport buffer. The transporter-mediated uptakerate was obtained after subtracting the uptake of thewater-injected oocytes from that of the cRNA-injectedoocytes. The inhibitory effect of luseogliozin wasexpressed as the percentage of the control.

Transporter inhibition experiments in cultured cells

The inhibition experiments were performed usingstably URAT1-expressing HEK293 cells andOAT4-expressing S2 cells (established from S2segments of mouse renal proximal tubules) in acontracted laboratory in a manner similar to thatdescribed previously [40]. The URAT1-expressingHEK293 cells were cultured in Dulbeccosmodied Eagles medium containing 10% FBS,penicillin, streptomycin, amphotericin B and 2

mM L-glutamine. The OAT4-expressing S2 cellswere cultured in RITC80-7 medium containing5% FBS, 10 g/l EGF, 0.08 units/ml insulin and10 mg/l transferrin, in a humidied 5% CO2 and95% air atmosphere at 37 C. For the inhibitionexperiment, three wells of cells for each conditionwere preincubated in transport buffer (Hanksbalanced salt solution) containing luseogliozinfor 15 min at 37 C. Uptake was initiated byreplacement with transport buffer containing[14C]UA (50 M) and the cells were incubated for2 min (URAT1) or 15 min (OAT4) at 37 C. The

cells were washed three times with ice-cold PBSand solubilized in 0.1 NNaOH for the quantica-tion of radioactivity. Part of the lysate was sub-jected to the protein measurement. The uptakerate (l/min/mg protein) was calculated bydividing the uptake amount by the initial concen-tration of the substrate in the transport buffer andthe protein amount of the cells. The transportermediated uptake rate and the inhibitory effectwere calculated in the same manner as that ofthe oocytes from the values of the control cells(transfected with the vector only) and the cRNA-

transfected cells.

Effects ofD-glucose on UA transport mediated byGLUT9 isoform 2

For the trans-stimulatory experiment on GLUT9 iso-form 2-mediated UA efux,Xenopus laevisoocyteswere injected with 0.125 ng of cRNA of GLUT9isoform 2 (Genbank NM_001001290.1) or waterand cultured at 18 C for 1 day. The UA-efux

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experiments were performed at 25 C using 810oocytes prepared from a single batch for eachgroup. Immediately after the injection of 50 nl of[14C]UA solution (1 mM) into the oocyte, the efuxreaction was individually initiated by replacement

with 100 l of transport buffer. After the reactionhad been allowed to occur for the designated time,transport buffer and oocyte were recovered. Theradioactivity in buffer and oocytes was quantiedto calculate the efux ratio of [14C]UA as thepercentage of the amount remaining within theoocytes or released into the buffer relative to the to-tal radioactivity. The transporter-mediated efuxrate was calculated by subtracting the values ofthe water-injected oocytes from those of the GLUT9isoform 2-expressing oocytes. To investigate theeffect on the UA efux, D-glucose was dissolved

in the transport buffer at concentrations of 5 or 10mM. The efux reaction was performed for 5 min.As a control study, transport buffer with 10 mMofL-glucose was used. Additionally, to investigatethe effect on the GLUT9 isoform 2-mediated UAefux, benzbromarone was dissolved in the trans-port buffer containing 10 mM D-glucose at a concen-tration of 100 M. For the stimulatory experimenton GLUT9 isoform 2-mediated UA uptake, theoocytes were injected with 25 ng of cRNA ofGLUT9 isoform 2 or water and cultured for 2 days.After the injection of 50 nl ofD-glucose or L-glucosesolution (100 mM each), the uptake of [14C]UA(10 M) was performed at 25 C for 15 minusing 18 oocytes for each group in transport bufferin which Na+ had been replaced with K+. Thetransporter-mediated uptake rate (l/15 min/oocyte)was calculated in the same manner as describedabove. For the inhibition experiment on the UAuptake by D-glucose, uptake was performed using1012 oocytes for each group in transport buffer with10 or 100 mMofD-glucose. As a control study, the ef-fect of 100 mM D-mannitol, 100 mM L-glucose and

transport buffer without any sugar were investigated.The other conditions were the same as the experimentfor the stimulatory effect ofD-glucose on UA uptake.

Uric acid uptake by SGLT2

The uptake experiments using stably SGLT2expressing CHO-K1 cells were performed asdescribed previously [10]. After preincubation ofthe SGLT2-transfected cells and the control cells in

Na+-free buffer (140 mM choline chloride, 2 mMKCl, 1 mMCaCl2, 1 mM MgCl2, 10 mMHEPES, 5mMTris, pH 7.4) at 37 C for 20 min, uptake wasinitiated by replacing Na+-free buffer with trans-port buffer (140 mMNaCl, 2 mMKCl, 1 mMCaCl2,

1 mM MgCl2, 10 mM HEPES, 5 mM Tris, pH 7.4)containing [14C]UA (9 M) or methyl--D-[U-14C]-glucopyranoside ([14C]-MG, 1 mM). Uptake wasallowed to occur for 5 min and was then terminatedby washing with ice-cold transport buffer. Theuptake rate (l/5 min/mg protein) was calculatedin the same manner as described above.

Statistical analyses

Data were expressed as the mean SEM or SD.Statistical signicance was determined using

Student

s t-test or Aspin-Welch

s t-test after ananalysis of variance when comparing two groupsand using Dunnetts test for multiple-groupcomparisons. A simple linear regression analysiswas performed using the standard method. Avalue ofp < 0.05 was considered signicant.

Results

SUA-lowering effect and uricosuric effect ofluseogliozin

The laboratory parameters from therst two clin-ical studies of luseogliozin in healthy subjectswere analysed [39]. Changes in the serum uricacid levels from the baseline values are shown inFigure 1. In the single dose study, the SUA levelwas signicantly decreased at doses higher than 1mg vs the placebo on day 1, and changes from thebaseline were0.66 to 1.59 mg/dl (Figure 1A).In the multiple dose study, the SUA level wasalso signicantly decreased at doses of 5 mg and10 mg vs the placebo on day 1, and the changes

were

1.39 and

1.33 mg/dl (Figure 1B). On day3, the changes in the SUA level were furtherincreased to1.76 and 1.71 mg/dl, which werecomparable to those observed on day 7.

Changes in the urinary excretion rate of uricacid (UEUA) and the CLUA from the baseline areshown in Figure 2. The UEUA was signicantlyincreased for all doses on day 1 vs the placebo inboth the single dose and the multiple dose studies.Changes from the baseline were 189268 mg/day

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and 243279 mg/day for the single dose and multipledose studies, respectively, on day 1 (Figure 2A, B).In the multiple dose study, the change in theUEUAfrom the baseline was the largest on day 1, with themagnitude of change decreasing on days 3 and 7(Figure 2B). On the other hand, the CLUA wasincreased on days 17 in both dose groups in themultiple dose study (Figure 2D).

Association of SUA with UEUA after administrationof luseogliozin

Changes in the SUA and UEUA levels from the base-line values in the three representative dose groups(1, 5 and 25 mg) are shown in Figure 3A, B, respec-tively. In the 1 mg dose group, the decrease in theSUA level was not signicant on day 1 and theUEUAwas increased on day 1 but had returned tothe baseline value on day 2. In the 25 mg group, amarked reduction in the SUA value continued until

day 4, while the increase in theUEUA

value contin-ued until at least day 2. In the 5 mg group, thechanges in the serum uric acid and UEUA levelswere intermediate to those for the previous twodoses. The correlation between the changes in theserum uric acid level and the UEUA relative tothe baseline on day 1 for all subjects (125 mg) isshown in Figure 3C. The change in the SUA levelshowed a strong negative correlation with thechange in theUEUA(r= 0.7672,p < 0.001).

Association of UEUAwith UEGLor plasmaconcentration of luseogliozin

The time courses of the increase inUEUA vs theplacebo group, the plasma concentration ofluseogliozin and the increase in the urinaryexcretion rate of glucose (UEGL) vs the placebogroup at the three representative doses are shownin Figure 4AC. The increase in the UEUA wasrelatively high at two time points (at 28 h and at1216 h post-dose, Figure 4A), whereas the plasmaconcentration of luseogliozin reached a maximumat 1 h post-dose and then decreased (Figure 4B).The increase in theUEGLshowed a similar transitionto that of theUEUA, since theUEGLincreased mark-edly at around 5 h and 13 h post-dose after foodintake (Figure 4C). Additionally, the correlation be-tween the changes in UEUAandUEGLvalues wascompared with the correlation between the changesinUEUAand the plasmaAUCvalues after a single

dose of luseogli

ozin (Figure 4D, E). The change inUEUAwas more strongly correlated with the changeinUEGL(r= 0.7875,p < 0.001, Figure 4D), than withtheAUC(r= 0.5707,p < 0.001, Figure 4E).

Effect of luseogliozin on transporters involved inrenal uric acid reabsorption

To determine whether luseogliozin has a directuricosuric effect on uric acid handling in the kidney,

Figure 1. Effect of luseogliozin on the serum uric acid (SUA) level. Changes in the SUA level from the baseline after a single dose(A,n = 314) and after multiple doses (B,n = 8) are shown. Data are mean SEM. **p < 0.01 vs placebo (0 mg) (Dunnetts test)





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the effect of luseogliozin on [14C]UA transportmediated by URAT1, GLUT9 isoform 1, OAT4 orOAT10 was examined using in vitro cell culturemodels expressing the respective transporter genes.Additionally, because SMCT1 transports monocar-boxylic acids, such as lactic acid and nicotinic acid,

which stimulate the exchange transport of uric acidvia URAT1 [43,44], the effect of luseogliozin onSMCT1-mediated [3H]nicotinic acid transport wasalso examined. Luseogliozin was found not to af-fect the activities of any of the transporters (Figure 5).

Uric acid transport by SGLT2

The study examined uric acid transport in CHO-K1cells stably expressing SGLT2 to test the hypothesis

thatUEUAshould be increased after treatment withSGLT2 inhibitors. The uptake of a known substrate,[14C]-MG, by SGLT2-expressing cells was signi-cantly greater than that by control cells (8.15 1.49vs 2.35 0.96 l/5 min/mg protein, mean SEM).On the other hand, the uptake of [14C]UA by

SGLT2-expressing cells and control cells were 1.73 0.44 l/5 min/mg protein and 2.12 0.75 l/5min/mg protein (mean SEM), respectively, sug-gesting that uric acid was not transported by SGLT2.

Trans-stimulatory effect ofD-glucose on uric acidtransport by GLUT9 isoform 2

Thetrans-stimulatory effect ofD-glucose on efuxof [14C]UA mediated by GLUT9 isoform 2 was

Figure 2. Effects of luseogliozin on the urinary excretion rate (UEUA) and the renal clearance (CLUA) of uric acid. Changes in UEUAandCLUAfrom the baseline after a single dose (A and C, n = 314) and after multiple doses (B and D, n = 8) are shown. Data aremean SEM. **p < 0.01 vs placebo (0 mg) (Dunnetts test)

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examined using oocytes expressing GLUT9 isoform2. The [14C]UA efux rate was clearly changed de-pending on the amount of cRNA of GLUT9 isoform2 injected into the oocytes at a range of 0.125 ng

(Figure 6A) and increased linearly over 15 min(Figure 6B). Based on these results, in order to moreclearly evaluate [14C]UA efux, oocytes injectedwith a smaller amount (0.25 ng) of cRNA in ashorter measurement time (5 min) compared with

the ordinary conditions were examined [31,41].The ef ux of [14C]UA injected into oocytesexpressing GLUT9 isoform 2 in the presence of10 mM D-glucose was signicantly higher than thatof 10 mM L-glucose (Figure 6C). Additionally, thestimulated [14C]UA efux by 10 mM of D-glucosewas decreased in the presence of 100 M ofbenzbromarone [2931] (Figure 6D). To conrmthe exchange transport of UA with D-glucose, thetrans-stimulatory effect of D-glucose on [14C]UAuptake was examined upon injection of 50 nl of100 mM of D-glucose solution into oocytes just

prior to [14C]UA uptake by the GLUT9 isoform2-expressing oocytes; uptake was indeed signi-cantly higher than that of 100 mM of L-glucose(Figure 6E).

Cis-inhibitory effect ofD-glucose on uric aciduptake by GLUT9 isoform 2

The cis-inhibitory effect of D-glucose on GLUT9isoform 2-mediated [14C]UA uptake was exam-ined at concentrations of 10 and 100 mM. A signif-icant difference was observed between the groups

of control (transport buffer) and 100 mM D-glucose(p < 0.05, Aspin-Welchs t-test), although no sig-nicant difference was observed as a result ofmultiple comparison (Dunnetts test) (Figure 7).

Discussion

The present study elucidated the mechanism of theSUA-lowering effect that is commonly observed in re-sponse to SGLT2 inhibitors [58] using luseogliozin

as a model drug.First, the study evaluated whether the change inthe serum uric acid level can be attributable to thechange in the UEUA using an SGLT2 inhibitor inclinical studies. When luseogliozin was adminis-tered to healthy subjects, a decrease in the SUAlevel and an increase in theUEUA were observed(Figures 1 and 2). After a single administration(125 mg), a negative correlation was observedbetween changes in the serum uric acid level and

Figure 3. Relationship between the serum uric acid (SUA) leveland the urinary excretion rate of uric acid (UEUA) after a singledose. The daily changes in the SUA level (A) and UEUA(B) from

the baseline inthe 1, 5 and 25 mg dosinggroups are shown. Dataare mean SEM. *p < 0.05, **p < 0.01 vs placebo (Dunnetts test,Studentst-test or Aspin-Welchst-test). (C) Correlation betweenthe changes in the SUA level and the UEUAon day 1 from the

baseline values in the single dose study





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theUEUA (Figure 3C). In addition, the increase inthe UEUA from the baseline value on day 1accounted for 33%

46%. Although this percentage

was greater than that of the decrease in the SUAlevel (8%25%), the extent of the increase in theUEUA was considered reasonable as an explanationfor the decrease in the SUA level, since the total uricacid in the body is approximately 1200 mg inhealthy subjects [45]. These observations suggestthat the SUA-lowering effect is due to an increasein theUEUA. In addition, the increase in theUEUAafter multiple dosing was marked on day 1 anddecreased on days 3 and 7 (Figure 2B), probably be-

cause of the decrease in the SUA level. TheCLUA

remained at a high level until day 7 (Figure 2D),suggesting that the SUA-lowering effect continued.

Second, the mechanism responsible for theuricosuric effect of an SGLT2 inhibitor was exam-ined. Previously, Skeithet al. reported that a non-selective SGLT inhibitor, phloridzin, andD-glucoseexerted a uricosuric effect that was stronger thanthat of mannitol after a bolus infusion in humans[11]. Hence, the effects of phloridzin andD-glucose

were estimated to be due not only to osmotic diure-sis, but also to an effect on uric acid handling in thekidney. Additionally, Knight et al. reported thatboth phloridzin and D-glucose inhibited uric acidreabsorption in rat proximal tubules [46], whilethe mechanisms underlying their uricosuric ef-fects were unclear. Based on our clinical studiesand these previous observations, it was postu-lated that the glucose level is directly related tothe SUA-lowering effect of SGLT2 inhibitors.When the dose of luseogliozin was changedfrom 1 mg to 25 mg, the time prole for theincrease in theUEUA was comparable to that for

the UEGL

, but not with that for the plasmaconcentration of luseogliozin (Figure 4AC).Additionally, theUEUAwas more strongly corre-lated with the UEGL than with the plasma AUCof luseogliozin (Figure 4D, E). Recent studieshave demonstrated clearly that a uricosuric effectof several drugs, such as benzbromarone, probene-cid, the angiotensin II receptor blocker, losartan,and salicylic acid, are due to the inhibition ofURAT1, which is expressed at the apical membrane

Figure 4. Comparison of the urinary excretion rate of uric acid (UEUA), the plasma concentration of luseogliozin and the urinaryexcretion rate of glucose (UEGL) in the single dose study. (AC) Time course proles of UEUA, the plasma concentration ofluseogliozin andUEGL. Data are mean SEM. (D) Relationship between UEUAand UEGL. (E) Relationship betweenUEUAand

AUC024hof plasma luseogliozin

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membrane [42,48], but its physiological role is un-known. Reportedly, D-glucose does not stimulateuric acid transport mediated by the GLUT9 isoform2 at a concentration of ~5 mM[30,49]. However, itwas hypothesized that the GLUT9 isoform 2 isstimulated by D-glucose at concentrations higherthan 5 mM, since it had not been examined at theseconcentrations. Additionally, the two isoforms dif-fer only in theNterminal [48] and their transportfunctions of uric acid and hexoses are similar[30,31]. In healthy humans, the average plasma glu-cose level is 55.5 mMand increases up to approxi-mately 10 mM after food intake [4]. Most of theplasma glucose is ltered at the glomerulus andthe ltrate is concentrated in the proximal tubule.

Because SGLT2 inhibitor blocks SGLT2 localizedat the S1 segment in the beginning of the proximaltubule, the glucose level in this part is likely to behigher than 5 mM.

To address this possibility, the study examinedthe effect of 5 mM and a higher concentration ofD-glucose on [14C]UA efux mediated by GLUT9isoform 2 using Xenopus oocytes expressing thetransporter. As a result, a signicant stimulation of[14C]UA efux by D-glucose was observed at a con-centration of 10 mM (Figure 6C). The contribution ofGLUT9 isoform 2 to this enhanced uric acid efuxwas conrmed by the inhibition of a high concen-tration of benzbromarone [2931] (Figure 6D). Inaddition, it was observed that D-glucose stimulated

Figure 6.Trans-stimulatory effect ofD-glucose on uric acid transport by GLUT9 isoform 2. Effect of the injected amount of cRNA of

GLUT9 isoform 2 into Xenopus oocytes on UA efux (n = 78) determined at 5 min and (B) time-course of UA efux from theoocytes injected with 0.25 ng of cRNA (closed circle) or water (open circle) (n= 48). (C) Trans-stimulatory effect of D-glucose(n= 510) and (D) cis -inhibitory effect of benzbromarone (Benz) (n= 710) on GLUT9 isoform 2-mediated [

14C]UA efux. (E)

Trans-stimulatory effect ofD-glucose on GLUT9 isoform 2-mediated [14

C]UA uptake (n= 1417). The efux and uptake of [14

C]UAwereevaluated using oocytes injected with0.25 ng and 25 ng ofcRNA, respectively. Data are mean SEM. *p< 0.05vs10 mM L-glucose(C, Dunnetts test) or vs 10 mM D-glucose (D, Aspin-Welchst-test), **p < 0.01 vs 100 mM L-glucose (E, Aspin-Welchst-test)

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uric acid transport by GLUT9 isoform 2 in the op-posite direction (Figure 6E). Our data are consis-tent with a previous study that showed anegative stimulatory effect of D-glucose on bothuric acid efux and uptake [30,49]. In ourin vitroexperiments, the stimulatory effect was not stablyreproducible when 25 ng cRNA/oocyte wasinjected. However, the stimulation effect becamereproducible when the efux activity of GLUT9was lowered by decreasing the injected amountof cRNA to 0.25 ng/oocyte. The observation sug-gests that the intracellularly injected uric acid wasrapidly efuxed by the GLUT9 isoform 2 when itis sufciently expressed by thein vitroexperiment.In addition, since GLUT9 is thought to be an efuxtransporter of anionic uric acid using a negativemembrane potential [30], uric acid uptake was

examined under degraded membrane potentialconditions. As a result, the basal uric acid uptakewas increased and a D-glucose-induced stimulationof uric acid efux was observed. Very recently,Kimura et al. reported the expression of GLUT9isoform 2 protein in the collecting ducts and thepossibility of its contribution to uric acid reabsorp-tion [50]. Urinary concentrations of glucose higherthan 100 mM were observed clinically after theadministration of luseogliozin at doses of> 3 mg

(data not shown). As a result, uric acid uptake byGLUT9 isoform 2-expressing oocytes was inhibitedby D-glucose at 100 mM(Figure 7). Based on theseresults, one can conclude that the increased concen-tration of glucose in the lumen by SGLT2 inhibitionstimulates uric acid excretion mediated by GLUT9isoform 2 or any other transporter(s) in the proxi-mal tubule and inhibits uric acid reabsorptionmediated by GLUT9 isoform 2 in the collecting duct(Figure 8).

The serum uric acid level is closely related tomarkers of insulin resistance [38]. Insulin has beensuggested to increase uric acid reabsorption withan increased Na+ reabsorption in the proximaltubule [34]. On the other hand, the serum uric acidlevel of patients with type 2 diabetes is known tobe lower than that of the normal population

[35,37,38], and the CLUA

of the patients is higherthan that of the normal population [37]. Moreover,the SUA reportedly increases and the UEUAdecreases after glycemic control for the treatmentof diabetes [51]. In addition, UEUA is induced byglycosuria after D-glucose infusion [11,52]. Untilnow, the decrease of SUA in patients with type 2diabetes has been explained by osmotic diuresiscaused by glucose [11,51], an alteration in theproximal tubule [51] and/or the effect of glucose

Figure 7. Cis-inhibitory effect of D-glucose on GLUT9 isoform2-mediated [

14C]UA uptake in oocytes injected with 25 ng of

cRNA (n = 8

12). Data are mean SEM. *p

sglt2 inhibitor lowers serum uric acid through alteration.pdf

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