alteration of the intestinal environment by lubiprostone...

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Alteration of the Intestinal Environment byLubiprostone Is Associated with Amelioration ofAdenine-Induced CKD

Eikan Mishima,*† Shinji Fukuda,‡§ Hisato Shima,* Akiyoshi Hirayama,‡ Yasutoshi Akiyama,*†

Yoichi Takeuchi,* Noriko N. Fukuda,‡ Takehiro Suzuki,* Chitose Suzuki,* Akinori Yuri,|

Koichi Kikuchi,¶ Yoshihisa Tomioka,| Sadayoshi Ito,* Tomoyoshi Soga,‡ and Takaaki Abe*¶**

*Division of Nephrology, Endocrinology, and Vascular Medicine and ¶Department of Clinical Biology and HormonalRegulation, Tohoku University Graduate School of Medicine, Sendai, Japan; †Tohoku Medical Megabank Organization,Tohoku University, Sendai, Japan; ‡Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan; §RIKEN Centerfor Integrative Medical Sciences, Yokohama, Japan; |Laboratory of Oncology, Pharmacy Practice and Sciences, TohokuUniversity Graduate School of Pharmaceutical Sciences, Sendai, Japan; and **Department of Medical Science, TohokuUniversity Graduate School of Biomedical Engineering, Sendai, Japan

ABSTRACTThe accumulation of uremic toxins is involved in the progression of CKD. Variousuremic toxins are derived from gut microbiota, and an imbalance of gut microbiotaor dysbiosis is related to renal failure. However, the pathophysiologic mechanismsunderlying the relationship between the gut microbiota and renal failure are stillobscure. Using an adenine-induced renal failure mouse model, we evaluated theeffects of the ClC-2 chloride channel activator lubiprostone (commonly used for thetreatment of constipation) onCKD.Oral administration of lubiprostone (500mg/kgperday) changed the fecal and intestinal properties inmice with renal failure. Additionally,lubiprostone treatment reduced the elevated BUN and protected against tubulointer-stitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNAgenes in the renal failure mice showed that lubiprostone treatment altered their mi-crobial composition, especially the recovery of the levels of the Lactobacillaceae familyand Prevotella genus, which were significantly reduced in the renal failure mice. Fur-thermore, capillary electrophoresis–mass spectrometry-based metabolome analysisshowed that lubiprostone treatment decreased the plasma level of uremic toxins, suchas indoxyl sulfate and hippurate, which are derived from gut microbiota, and a morerecently discovered uremic toxin, trans-aconitate. These results suggest thatlubiprostone ameliorates the progression of CKD and the accumulation of uremictoxins by improving the gut microbiota and intestinal environment.

J Am Soc Nephrol 26: 1787–1794, 2015. doi: 10.1681/ASN.2014060530

CKD is a global health problem thatcarries a substantial risk for ESRD, car-diovascular disease, and death.1 In CKD,the accumulation of uremic toxins ac-celerates the progression of CKD andhence, mortality.2,3 Therefore, reducingthe accumulation of uremic toxins is im-portant for the protection against andamelioration of renal damage.

Recent evidence has suggested thatalterations in the gut microbiota arelinked to various diseases conditions,including obesity and diabetes as wellas cardiovascular4,5 and kidney dis-eases.6,7 Because various uremic solutes,such as indoxyl sulfate, are derived fromgut microbial metabolism, the gut mi-crobial status affects the accumulation of

uremic toxins.8 Furthermore, it has beenreported that the gut microbiota compo-sition is changed for the worse in CKD,6

and the gut environment is associatedwith both the etiology and progressionof CKD. However, the mechanistic linkbetween the gut and CKD remains poorlyunderstood.

Lubiprostone is a synthetic bicyclicfatty acid derivative of prostaglandin E1that is clinically used for the treatment ofchronic constipation.9 Lubiprostoneactivates the ClC-2 chloride channel,resulting in an enhancement of the in-testinal luminal Cl2 secretion and watermotility, which effects are responsiblefor its laxatives properties.10 In general,

Received June 2, 2014. Accepted October 30,2014.

E.M. and S.F. contributed equally to this work.

Published online ahead of print. Publication dateavailable at www.jasn.org.

Correspondence: Prof. Takaaki Abe, Division ofMedical Science, Tohoku University GraduateSchool of Biomedical Engineering, Department ofClinical Biology and Hormonal Regulation, TohokuUniversity Graduate School of Medicine, Sendai980-8574, Japan. Email: [email protected]

Copyright © 2015 by the American Society ofNephrology

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chronic constipation, which is frequentin patients with CKD, worsens the intes-tinal environment and alters the com-position of the gut microbiota.11,12 Weexamined the beneficial effects oflubiprostone on CKD using an adenine-induced renal failure (RF) mouse model.Lubiprostone was found to exert a reno-protective effect on the progression ofCKD with a reduction in the plasma con-centration of uremic toxins and improve-ment in the gut microbiota population.These findings suggest a potential ther-apeutic approach to CKD on the basis ofthe improvement of the intestinal envi-ronment.

We examined the effect of lubiprostoneon uremic RF using an adenine-inducedRF mouse model13 (Supplemental Fig-ure 1). RF was developed by 6-week oraladministration of adenine into normalmice, which were then treated with 50or 500 mg/kg per day lubiprostone(RF+Lub50 and RF+Lub500, respec-tively). RF mice exhibited reduced foodintake, body weight loss, abnormal poly-uria, and anemia compared with controlmice (Supplemental Table 1). Bodyweight and food intake were comparableamong the RF, RF+Lub50, and RF+Lub500groups, but the serum sodium levelwas significantly reduced in both of thelubiprostone-treated RF groups (148.3and 146.7 mEq/L in RF+Lub50 andRF+Lub500 groups, respectively) com-pared with the RF group (153.0 mEq/L).Concerning the fecal and intestinal ef-fects, the RF+Lub500 group exhibited asignificantly higher level of each fecalsample weight and a lower amount ofresidual feces in the colon than the RFgroup (Figure 1, A and 1B). In addition,slightly soft feces were observed in theRF+Lub500 group because of the in-crease in the small intestinal bowel fluid(Figure 1C) and accelerated intestinaltransit (Figure 1D). However, diarrheawas not observed in either the RF+Lub50or RF+Lu500 group. The pH of the cecalfluid was comparable among the groups(pH was 6.560.33, 6.660.34, and6.560.29 in control, RF, and RF+Lub500groups, respectively). ByMasson’s trichromestaining (MTS) of the colon, dense col-lagen deposition in the lamina propria

was detected in the RF group as re-ported.14 This collagen deposition inthe colon was suppressed in the RF+Lub500 group compared with the RFgroup (Figure 1E).

We next examined the effect oflubiprostone on renal function and histo-logicchanges in theRFmice.TheBUNlevelwas 4.1-fold higher in the RF group than inthe control group, whereas it was signifi-cantly suppressed in theRF+Lub500 group(Figure 2A). In addition, the decrease inthe cortical tubular area in the RF groupwas slightly but significantly recovered inthe RF+Lub500 group (Figure 2B), al-though the adenine-derived cast formationwas also observed in theRF+Lub500 groupin a similar manner to the RF group (Fig-ure 2C, periodic acid–Schiff staining). Incontrast, the fibrotic area and interstitial

collagen deposition in the damaged kid-neys were decreased in the RF+Lub500group compared with the RF group(Figure 2C).

We further evaluated renal fibrosisand inflammation by immunohistochem-istry and quantitative PCR. By immuno-histochemical analysis, the RF+Lub500group exhibited a decreased number ofmacrophages and myofibroblasts com-pared with the RF group, which wasshownby anti-F4/80 and anti–a-smoothmuscle actin staining (Figure 2D).Quantitative PCR indicated a significantreduction in the expression of renalfibrosis-related genes (Acta2, Tgfb1,Col1a1, and Col3a1) and inflammatorycytokines (Tnfa, Pai-1, and Ccl2) in thekidney of the RF+Lub500 group com-pared with the RF group (Figure 3). The

Figure 1. Effect of lubiprostone on fecal and intestinal characteristics in uremic mice. (A)Fecal shape andweight per pellet. Fecal weight per pelletwas calculated as (fecal number)/(total fecal wet weight). n=6–7 for each group. (B) Residual fecal number in the colon. n=6–7.(C) Representative small intestinal images of RF and RF+Lub500 groups. (D) Intestinaltransit analysis after lubiprostone administration in RF mice. The arrowheads denote theleading edge of trypan blue. *P,0.05 between the indicated groups. n=3. Scale bar, 2 cm.(E) Representative histologic images of the colon. MTS-stained cross-section of the coloncontaining a fecal pellet. The arrow indicates lamina propria. P,0.05 between the in-dicated groups. Cont, control. Scale bar, 100 mm.

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expression of Il-6 mRNA was not signifi-cant but tended to be reduced in the RF+Lub500 group. These results suggestedthat the treatmentwith lubiprostone ame-liorated the progression of renal fibrosisand local inflammation in the RF mousemodel.

To elucidate the mechanism of therenoprotectiveeffect inducedbylubiprostone

treatment, we investigated the effect oflubiprostone on the gut microbiota inRF mice. Gut microbiome analysis ofbacterial 16S rRNA genes followed byoperational taxonomic unit and un-weighted Unifrac distance analysis indi-cated that the gut microbial compositioncould be roughly categorized into threegroups (i.e., the control, RF, and RF+Lub500

groups) (Figure 4A). In addition, the di-versity analysis showed that the decreasedmicrobial diversity in the RF group be-came more similar to the control groupby lubiprostone treatment (Figure 4A).Consistent with this, microbial populationanalysis showed that aminor populationof gut microbiota in each group was differ-ent (Figure 4B). At the genus level, quanti-tative analysis confirmed a significantincrease inAllobaculum, Bfidobacterium,Clostridium, and Parabacteroides in boththe RF and RF+Lub500 groups (Figure4C). Interestingly, an unclassified Lacto-bacillaceae family, a Prevotella genus, anunclassified Clostridia class, and an un-classifiedmitochondria family were signif-icantly decreased in the RF group, but thisdecrease was not found in the RF+Lub500group (Figure 4C). Furthermore, the sig-nificant increase in the Lactobacillus andTuricibacter genuses in the RF group wasalso abrogated by lubiprostone treatment,suggesting that lubiprostone is able toameliorate gut microbiota imbalances inRF mice.

To exclude the possibility that theadenine diet per se induced the alterationof gut microbiota, we performed a fecalmicrobiome analysis during adeninefeeding (Supplemental Figure 2A). Thecomposition of the microbiota did notobviously change 1 and 2 weeks after ad-enine feeding. BUN began to signifi-cantly increase after the 2-week adeninediet (Supplemental Figure 2B). A slightalteration of the microbiota was ob-served after 4 weeks and evident after 6weeks of the adenine diet. Simulta-neously, BUN was markedly increasedafter 4 and 6 weeks of the adenine diet.

These results suggest that the adeninediet per se had a little influence on the gutmicrobiota and that the alteration of thegut microbiota in adenine-induced RFwas mainly dependent on the RF state.

We next performed capillary electro-phoresis with electrospray ionizationtime-of-fl ight mass spectrometry(CE-TOFMS)-based metabolomeanalysis of the plasma in the control,RF, andRF+Lub500groups.The increasedlevels of the well known uremic solutesindoxyl sulfate and hippurate in the RFgroup were significantly suppressed in

Figure 2. Effect of lubiprostone on renal fibrosis and inflammation in the uremic mousekidney. (A) BUNconcentration inmice.n=6–7 for eachgroup. (B)Morphometric analysis of thepercentage of the cortical tubular area and whole-kidney images ofMTS. The cortical tubulararea was calculated on the basis of the MTS image. n=6–7. Scale bar, 1 cm. *P,0.05 com-paredwith theRFgroup; **P,0.01comparedwith theRFgroup. (C)Representativehistologicimages of periodic acid–Schiff- (PAS-), MTS-, and picrosirius red-stained kidney. The arrow-heads denote adenine-derived casts. Scale bar, 100 mm. (D) Immunostaining of a-smoothmuscle actin (a-SMA) and F4/80 in kidney sections. Cont, control. Scale bar, 100 mm.

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the RF+Lub500 group (Figure 5A). Inaddition to these uremic solutes, higherplasma concentrations of certain anionicmetabolites in theRF group, such as trans-aconitate, propionate, and cholate, weresignificantly suppressed by lubiprostonetreatment (Figure 5A). Furthermore,most of the tricarboxylic acid cycle-relatedcomponents (citrate, cis-aconitate, iso-citrate, fumarate, and malate) were sig-nificantly increased in both the RF andRF+Lub500 groups compared with thecontrol group. Among the cationic solutes,the amino acids tryptophan, glutamate,inosine, and betaine were significantlydecreased in both RF and RF+Lub500groups, whereas the elevated concentra-tion of g-aminobutyric acid in RFwas sig-nificantly decreased in the RF+Lub500group compared with the RF group. (Fig-ure 5B). The plasma acetate and p-cresylsulfate concentrationswere alsomeasured(Supplemental Figure 3). The plasma ac-etate concentration was comparable

among these groups. The plasma p-cresylsulfate level was significantly increased inboth the RF and RF+Lub500 groups com-pared with the control group but notchanged between the RF and RF+Lub500groups.

In CKD, the intestinal environment isreported to be adversely altered.15,16 Thepotential exacerbating factors includethe retention of uremic toxins, intestinalischemia, intestinal transit time pro-longed by constipation, decreased intes-tinal fluid secretion, and malnutrition ofthe gut lining with evident atrophy.8,16

Such a poor condition of the gut is sug-gested to be involved in the etiology andprogression of CKD because of an im-balance in the gut microbiota, leading toan accumulation of uremic toxins andbacterial translocation.16

Inthisstudy,weshowedthatlubiprostonetreatment ameliorated the accumulationof uremic toxins andmitigated the renalfibrosis and inflammation that play a

major role in the progression of renaldamage.17 Lubiprostone actively inducedbowel fluid secretion by activating Cl2

channels, leading to a reduction in the in-testinal transit time and changes in thefecal characteristics through alteringthe gut environment. This gut micro-biome analysis showed that the relativeabundance of the Lactobacillaceae familyand Prevotella genus was significantlydecreased in the RF group, which is con-sistent with a previous report.6 Further-more, our findings show that lubiprostonetreatment of RF mice ameliorated the de-crease in the Lactobacillaceae family andPrevotella genus. This increased abun-dance of the Lactobacillaceae family bylubiprostone treatment has also beenreported in a previous study using a cysticfibrosis model mice.18

It has also been reported that the gutmicrobiota largely contributes to theproduction of various uremic solutes, suchas indole derivatives (e.g., indoxyl sulfate)and glycine conjugates (e.g., hippurate)19;therefore, dysbiosis might be related to theaccumulation of uremic toxins. In thisstudy, oral administration of lubiprostonereduced the plasma concentration of in-doxyl sulfate and hippurate as well astrans-aconitate. Indoxyl sulfate is associatedwith the progression of RF through theexacerbation of oxidative stress, inflam-mation, and fibrosis.20 Trans-aconitateis also known as a uremic toxin and a re-cently identified biomarker for predictingthe onset of renal damage.21 Therefore,the alteration of the gut microbiota in-duced by treatment with lubiprostone inRF mice is suggested to have caused theimprovement of the plasma uremic sol-ute levels, which in turn, led to the ame-lioration of CKD.

Lactobacillaceae and Prevotella, whichwere increased by the lubiprostone treat-ment, are saccharolytic bacteria.Indoles, a precursor of indole deriva-tives, are produced as a result of proteinfermentation by colonic microbiota.8

Therefore, we suppose that lubiprostonemay promote the growth of saccharolyticbacteria in RF at the expense of the otherproteolytic bacteria, resulting in the re-duction of some uremic toxins (e.g., in-doxyl sulfate).

Figure 3. Effects of lubiprostone on renal gene expression in uremic mice. Relative tran-script levelsofgenes in (A) renal inflammationand (B)fibrosisweremeasuredusing real-timePCR. The names in parentheses indicate the coding protein: Tnfa (TNF-a), Il6 (IL-6), Pai-1(PAI-1), Ccl2 (MCP-1), Col1a1 (type I collagen-a1), Col3a1 (type III collagen-a1), Tgfb1(TGF-b), and Acta2 (a-smooth muscle actin). The expression levels were first normalized tothose of Gapdh and then further normalized to the levels in the kidney from the controlmice. n=6–7 for each group. Cont, control. *P,0.05 versus the RF group; **P,0.01 versusthe RF group.




In addition, it has been reportedthat a prolonged intestinal transit timepromotes the conversion of amino acidsinto uremic wastes through microbialfermentation.11 Thus, a reduction of theintestinal transit time by lubiprostonetreatment may also be involved in the

reduction in uremic solutes and theamelioration of CKD symptoms.

Intestinal mucosal barrier functionsare impaired in CKD.22 This impairmentleads to a high intestinal epithelial per-meability, resulting in bacterial translo-cations from the gut lumen into the

blood stream, and this increased bacterialtranslocation is reported to be a source ofmicroinflammation in CKD.23 Recently,Moeser et al.24 reported that lubiprostoneexerts a reparative effect on the functionof the intestinal mucosal barrier againstischemic intestinal damage. In addition,lubiprostone treatment stimulated theintestinal secretion of mucin,25 whichworks as the first line of defense of thegastrointestinal mucosal barrier. There-fore, the preventive effect of lubiprostonetreatment in RF mice might be throughupregulation of the mucosal barrierfunction.

Vaziri et al.26 have reported that theuremia-induced disruption of the intes-tinal barrier is mediated by urea influxinto the gastrointestinal tract. Influxurea is converted by microbial ureaseto ammonia and ammonium hydroxide.These caustic compounds can disruptthe intestinal barrier function. Further-more, the uremia-induced alteration ofthe intestinal microbiota is driven by theinflux of urea into the gastrointestinaltract.14 Therefore, some effects oflubiprostone on the urea influx into thegastrointestinal tract may be postulated.Additional experiments are necessary toclarify this issue.

A recent study has shown expansionof bacteria families possessing urease,uricase, and indole- and p-cresol–formingenzymes and reduction of butyrate-producing bacteria in patients withESRD. Wong et al.27 have shown suchdysbiosis in patients with ESRD usingin silico tests. In accordance with the insilico results,27 these data show the reduc-tion of bacteria families possessing butyrate-producing enzymes (Lactobaillaceae andPrevotellaceae) in the RF mice, and thereduction was recovered by lubiprostonetreatment (Supplemental Figure 4).However, the reported bacterial familiesthat possess urease, uricase, and indole-and p-cresol–forming enzymes27 en-riched in patients with ESRD were notsignificantly changed in the RF mice ornot detected in any groups (SupplementalFigure 4). Additionally, Bifidobacteriumwas scarce in the normal mice and in-creased in the RFmice, although previousreports showed that Bifidobacterium is

Figure 4. Effects of lubiprostone on gut microbiota in uremic mice. (A) Unweightedunifrac distance analysis (upper panel) and diversity of bacterial species as indi-cated by Chao1 rarefaction measure (lower panel) of gut microbiota in the control,RF, and RF+Lub500 groups. n=6. (B) Relative abundance of microbiota on the basisof the average number of each subfamily at the order, family, and genus levels.The major subfamilies are indicated on the right. n=6. Cont, control. (C) The pro-portional change of each subfamily at the genus level. Genera displaying a significantchange among the three groups are shown (Steel–Dwass). The y axis indicatesthe abundance of each microbe (percentage). C, control; RF+L, RF treated with500 mg/kg per day lubiprostone. *P,0.05 versus the control group; §P,0.05 versusthe RF group.






present under normal conditions anddecreases under RF states in rat andhuman.28,29 We suppose that such

discrepancies result from the differencesamong mammalian species. The bacte-rial families that contributed to the urease

or indole-producing activities in RF micemay differ from those of human ESRD.Also, the contribution of Bifidobacterium

Figure 5. Effects of lubiprostone on the plasma uremic solutes analyzed by CE-TOFMS–basedmetabolome analysis in uremicmice. Eachof the plasma concentrations was measured by CE-TOFMS. (A) Anionic and (B) cationic solutes. The y axis indicates the micromolarconcentration. n=6. C, control; GABA, g-aminobutyric acid; RF+L, RF treated with 500 mg/kg per day lubiprostone. *P,0.05 versus thecontrol group; §P,0.05 versus the RF group (ANOVA).



in the normal microbiota of mice is pre-sumably low, and the increase of theBifidobacterium population in RF is possi-bly related to the uremic dysbiosis in mice.In support of this idea, Bifidobacteriumwasalso undetectable among gut microbiotaof another mouse strain, BALB/c mice(S.F. et al., unpublished data).

In conclusion, lubiprostone treatmentis a potentially useful therapeutic inter-vention against the progression of CKDby improving the gut environment andreducing uremic toxins. These findingsprovide supporting evidence for the gut–kidney axis in CKD.

CONCISE METHODS

Animal StudiesAll animal experiments were approved by the

Animal Committee of the Tohoku University

School ofMedicine.Male C57BL/6mice were

fed a normal diet (type CE-2; Clea Japan). At

age 7weeks,micewere divided randomly into

the control and adenine groups. For the

control group, thenormaldietwas continued.

For the adenine group, a CE-2 diet containing

0.2% adenine (Wako) was given for 6 weeks.

After 6 weeks of adenine feeding, the food

was switched to the normal diet, and then,

lubiprostone (50 or 500 mg/kg; Abbott, Japan)

or saline in a volume of 100 ml was adminis-

tered to the mice by daily gavage at night for

12 days. At the end of study, the mice were

euthanized, after which blood, cecal fluid,

and tissues were obtained. One day before

euthanasia, mice were placed in an individual

metabolic cage to measure the water and food

intake and collect urine and feces. BUN and

biochemical parameters were assessed using a

blood analyzer (i-STAT; Fuso Pharmaceutical

Industries). BP was measured by the tail-cuff

method using model MK-2000 (Muromachi

Kikai). The cecal fluid pH was measured by a

pH meter (LAQUAtwin; HORIBA).

Intestinal Transit AnalysesThe measurement of gastrointestinal transit

was performed as described previously.30

Briefly, adenine-induced RF mice were fasted

for 20 hours with free access to water. Mice

were treated with either vehicle or lubiprostone

(50 or 500 mg/kg) orally, and 1 hour later, they

were given an oral gavage of 150 ml 0.5%

trypan blue solution. The mice were eutha-

nized 30 minutes after administration of the

solution, and the small intestine from the je-

junum to the cecum was dissected. The dis-

tance traveled by the leading edge of the small

intestine and the percentage of intestinal tran-

sit for each animal were calculated as the per-

centage of transit (trypan blue distance)/

(small intestinal length)3100.

Histology and MorphometricAnalysesTissues were fixed in 10% neutral buffered

formalin and embedded in paraffin. Kidney

sections were stained with hematoxylin and

eosin, periodic acid–Schiff,MTS, andpicrosirius

red. For analysis of the tubular area, the per-

centage of the tubular area in the cortex was

evaluated with MTS-stained kidney sections

using the National Institutes of Health

ImageJ analysis software. For immunohisto-

chemistry, sections were immunolabeled

using anti–a-smooth muscle actin (DAKO)

and anti-F4/80 (Serotec) antibodies.

Quantitative PCR AnalysesWhole kidneys were homogenized in TRIzol

reagent (Invitrogen) and extracted according

to the manufacturer’s directions. cDNA syn-

thesis was performed using a transcriptor

first-strand cDNA synthesis kit (Roche).

The primers purchased from Applied Biosys-

tems are shown in Supplemental Table 2.

Microbiome AnalysesThe genomic DNA of gut microbiota was

extracted frommurine feces, and 454-barcode

pyrosequencing of microbial 16S rRNA genes

was performed as described previously.31,32

Briefly, microbial genomic DNAwas extracted

using a phenol–chloroform standard protocol

with vigorous shaking with 0.1-mm zirconia/

silica beads. The V1–V2 region of the 16S

rRNA gene was amplified and subjected to

454 GS JUNIOR pyrosequencing (Roche).

The 16s rRNA reads were analyzed using

QIIME and the RDP classifier. The microbiome

data have been deposited in the DDBJ database

(http://getentry.ddbj.nig.ac.jp/) under acces-

sion number DRA002254. Detailed methods

are described in Supplemental Material.

CE-TOFMS MeasurementAquantitative analysis of chargedmetabolites

by CE-TOFMS was performed as described

previously.33,34 CE-TOFMS experiments

were performed using the Agilent CE System

(Agilent Technologies), the Agilent G3250AA

LC/MSD TOF System (Agilent Technolo-

gies), the Agilent 1100 Series Binary HPLC

Pump, the G1603A Agilent CE-MS adapter

(Agilent Technologies), and the G1607A

Agilent CE-ESI-MSSprayerKit.Detailedmeth-

ods are described in Supplemental Material.

Statistical AnalysesData are presented as the means6SD. Statis-

tical analysis was evaluated by the unpaired t

test as well as ANOVA or Steel–Dwass multi-

ple comparison procedures. Values of

P,0.05 were considered to be statistically

significant.

ACKNOWLEDGMENTS

This work was supported, in part, by JSPS

Grants-in-Aid forScientificResearch (KAKENHI)

Grants 26860624 (to E.M.), 24380072 (to S.F.),

and 23390033 (to T.A.), the Japan Kidney

Foundation, and theMiyagi Kidney Foundation.

DISCLOSURESNone.

REFERENCES

1. Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S,Plattner B, Saran R, Wang AY, Yang CW:Chronic kidney disease: Global dimension andperspectives. Lancet 382: 260–272, 2013

2. Vanholder R, Baurmeister U, Brunet P, CohenG,GlorieuxG, Jankowski J; EuropeanUremic ToxinWorkGroup: Abench to bedside viewof uremictoxins. J Am Soc Nephrol 19: 863–870, 2008

3. Niwa T, Nomura T, Sugiyama S, Miyazaki T,Tsukushi S, Tsutsui S: The protein metabolitehypothesis, a model for the progression ofrenal failure: An oral adsorbent lowers in-doxyl sulfate levels in undialyzed uremic pa-tients. Kidney Int Suppl 62: S23–S28, 1997

4. Tremaroli V, Bäckhed F: Functional inter-actions between the gut microbiota and hostmetabolism. Nature 489: 242–249, 2012

5. Fukuda S, Ohno H: Gut microbiome andmetabolic diseases. Semin Immunopathol

36: 103–114, 20146. Vaziri ND, Wong J, Pahl M, Piceno YM, Yuan

J, DeSantis TZ, Ni Z, Nguyen TH, AndersenGL: Chronic kidney disease alters intestinalmicrobial flora. Kidney Int 83: 308–315, 2013




http://getentry.ddbj.nig.ac.jp/



7. Ramezani A, Raj DS: The gut microbiome,kidney disease, and targeted interventions.J Am Soc Nephrol 25: 657–670, 2014

8. Meyer TW, Hostetter TH: Uremic solutesfrom colon microbes. Kidney Int 81: 949–954, 2012

9. Lacy BE, Levy LC: Lubiprostone: A chloridechannel activator. J Clin Gastroenterol 41:345–351, 2007

10. Fei G, Wang YZ, Liu S, Hu HZ, Wang GD, QuMH, Wang XY, Xia Y, Sun X, Bohn LM, CookeHJ,Wood JD: Stimulation ofmucosal secretionby lubiprostone (SPI-0211) in guinea pig smallintestine and colon. Am J Physiol GastrointestLiver Physiol 296: G823–G832, 2009

11. Stephen AM, Wiggins HS, Cummings JH:Effect of changing transit time on colonicmicrobial metabolism in man. Gut 28: 601–609, 1987

12. Khalif IL, Quigley EM, Konovitch EA,Maximova ID: Alterations in the colonic floraand intestinal permeability and evidence ofimmune activation in chronic constipation.Dig Liver Dis 37: 838–849, 2005

13. Tanaka T, Doi K, Maeda-Mamiya R, NegishiK, Portilla D, Sugaya T, Fujita T, Noiri E: Uri-nary L-type fatty acid-binding protein canreflect renal tubulointerstitial injury. Am JPathol 174: 1203–1211, 2009

14. Vaziri ND: CKD impairs barrier function andalters microbial flora of the intestine: A majorlink to inflammation and uremic toxicity.CurrOpin Nephrol Hypertens 21: 587–592, 2012

15. Evenepoel P, Meijers BK, Bammens BR,Verbeke K: Uremic toxins originating fromcolonic microbial metabolism. Kidney IntSuppl 114: S12–S19, 2009

16. Anders HJ, Andersen K, Stecher B: The in-testinal microbiota, a leaky gut, and abnor-mal immunity in kidney disease. Kidney Int83: 1010–1016, 2013

17. Declèves AE, Sharma K: Novel targets ofantifibrotic and anti-inflammatory treatmentin CKD. Nat Rev Nephrol 10: 257–267, 2014

18. Musch MW, Wang Y, Claud EC, Chang EB:Lubiprostone decreases mouse colonic innermucus layer thickness and alters intestinalmicrobiota. Dig Dis Sci 58: 668–677, 2013

19. Wikoff WR, Anfora AT, Liu J, Schultz PG,Lesley SA, Peters EC, Siuzdak G: Metab-

olomics analysis reveals large effects of gutmicroflora on mammalian blood metabo-lites. Proc Natl Acad Sci U S A 106: 3698–3703, 2009

20. Niwa T: Indoxyl sulfate is a nephro-vasculartoxin. J Ren Nutr 20[Suppl]: S2–S6, 2010

21. Toyohara T, Suzuki T, Morimoto R, AkiyamaY, Souma T, Shiwaku HO, Takeuchi Y,Mishima E, Abe M, Tanemoto M, Masuda S,Kawano H, Maemura K, Nakayama M, SatoH, Mikkaichi T, Yamaguchi H, Fukui S,Fukumoto Y, Shimokawa H, Inui K, TerasakiT, Goto J, Ito S, Hishinuma T, Rubera I, TaucM, Fujii-Kuriyama Y, Yabuuchi H, MoriyamaY, Soga T, Abe T: SLCO4C1 transportereliminates uremic toxins and attenuates hy-pertension and renal inflammation. J Am SocNephrol 20: 2546–2555, 2009

22. Vaziri ND, Yuan J, Nazertehrani S, Ni Z, Liu S:Chronic kidney disease causes disruption ofgastric and small intestinal epithelial tightjunction. Am J Nephrol 38: 99–103, 2013

23. Wang F, Jiang H, Shi K, Ren Y, Zhang P,Cheng S: Gut bacterial translocation is asso-ciated with microinflammation in end-stagerenal disease patients.Nephrology (Carlton)17: 733–738, 2012

24. Moeser AJ, Nighot PK, Roerig B, Ueno R,Blikslager AT: Comparison of the chloridechannel activator lubiprostone and the orallaxative Polyethylene Glycol 3350 on muco-sal barrier repair in ischemic-injured porcineintestine. World J Gastroenterol 14: 6012–6017, 2008

25. De Lisle RC: Lubiprostone stimulates smallintestinal mucin release. BMC Gastroenterol12: 156, 2012

26. Vaziri ND, Yuan J, Norris K: Role of urea inintestinal barrier dysfunction and disruptionof epithelial tight junction in chronic kidneydisease. Am J Nephrol 37: 1–6, 2013

27. Wong J, Piceno YM, Desantis TZ, Pahl M,Andersen GL, Vaziri ND: Expansion ofurease- and uricase-containing, indole- andp-cresol-forming and contraction of short-chain fatty acid-producing intestinal micro-biota in ESRD. Am J Nephrol 39: 230–237,2014

28. Wang F, Zhang P, Jiang H, Cheng S: Gutbacterial translocation contributes to micro-

inflammation in experimental uremia. DigDis Sci 57: 2856–2862, 2012

29. Wang IK, Lai HC, YuCJ, Liang CC, ChangCT,Kuo HL, Yang YF, Lin CC, Lin HH, Liu YL,Chang YC, Wu YY, Chen CH, Li CY, ChuangFR, HuangCC, Lin CH, Lin HC: Real-time PCRanalysis of the intestinal microbiotas in peri-toneal dialysis patients. Appl Environ Mi-crobiol 78: 1107–1112, 2012

30. Kakino M, Izuta H, Ito T, Tsuruma K, Araki Y,Shimazawa M, Oyama M, Iinuma M, Hara H:Agarwood induced laxative effects via ace-tylcholine receptors on loperamide-inducedconstipation in mice. Biosci Biotechnol Bio-chem 74: 1550–1555, 2010

31. Kim SW, SudaW, Kim S,Oshima K, Fukuda S,Ohno H, Morita H, Hattori M: Robustness ofgut microbiota of healthy adults in responseto probiotic intervention revealed by high-throughput pyrosequencing. DNA Res 20:241–253, 2013

32. Furusawa Y, Obata Y, Fukuda S, Endo TA,NakatoG, Takahashi D, Nakanishi Y, UetakeC,Kato K, Kato T, Takahashi M, Fukuda NN,Murakami S, Miyauchi E, Hino S, Atarashi K,Onawa S, Fujimura Y, Lockett T, Clarke JM,Topping DL, Tomita M, Hori S, Ohara O,Morita T, KosekiH,Kikuchi J,HondaK,HaseK,Ohno H: Commensal microbe-derived butyrateinduces the differentiation of colonic regulatoryT cells.Nature 504: 446–450, 2013

33. Soga T,Ohashi Y, Ueno Y, NaraokaH, TomitaM, Nishioka T: Quantitative metabolomeanalysis using capillary electrophoresis massspectrometry. J Proteome Res 2: 488–494,2003

34. Akiyama Y, Takeuchi Y, Kikuchi K, Mishima E,Yamamoto Y, Suzuki C, Toyohara T, Suzuki T,Hozawa A, Ito S, Soga T, Abe T: A metab-olomic approach to clarifying the effect ofAST-120 on 5/6 nephrectomized rats bycapillary electrophoresis with mass spec-trometry (CE-MS). Toxins (Basel) 4: 1309–1322, 2012

This article contains supplemental material onlineat http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014060530/-/DCSupplemental.





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