Research ArticleFebuxostat Attenuates Renal Damage besides ExertingHypouricemic Effect in Streptozotocin-Induced Diabetic Rats

Jianmin Ran12 Gang Xu12 HuixuanMa1 Hailing Xu1 Yan Liu23 Rongshao Tan2

Ping Zhu12 Jun Song4 and Gancheng Lao12

1Department of Endocrinology Guangzhou Red Cross Hospital Medical School of Jinan University No 396 Tong Fu Zhong RoadGuangzhou China2Guangzhou Institute of Disease-Oriented Nutritional Research Guangzhou Red Cross Hospital Medical School of Jinan UniversityNo 396 Tong Fu Zhong Road Guangzhou China3Department of Nephrology Guangzhou Red Cross Hospital Medical School of Jinan University No 396 Tong Fu Zhong RoadGuangzhou China4Southern Medical University No 1023-1063 Southern Sha Tai Road Guangzhou China

Correspondence should be addressed to Jianmin Ran ranjmmsncom

Received 3 March 2017 Revised 20 March 2017 Accepted 27 March 2017 Published 19 April 2017

Academic Editor Franca Anglani

Copyright copy 2017 Jianmin Ran et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aim In this study we aimed to investigate the effects of febuxostat a novel inhibitor of xanthine oxidase (XO) on renal damagein streptozotocin- (STZ-) induced diabetic rats Methods Diabetes was induced by the intraperitoneal injection of STZ in maleSprague-Dawley rats Sham-injected rats served as controls The control and diabetic rats were treated with and without febuxostatfor 8 weeks respectively Fasting blood and 24-h urine samples were collected every 4 weeks Rat livers were extracted for detectinggene expression content and bioactivity of XO Results Diabetic rats showed significantly increased serum uric acid (SUA) serumcreatinine (SCr) and urea nitrogen (BUN) levels Daily urinary albumin (UAE) uric acid (UUA) and creatinine (UCr) excretionwere also significantly increased in these rats In diabetic rats at week 8 febuxostat decreased SUA by 189 while UAA wasincreased by 520 However UCr and urinary urea nitrogen (UUN) levels remained unchanged while SCr and BUN levelsdecreased by gt30 in these rats Although hepatic gene expression content and activity of XO increased significantly in diabeticrats febuxostat only slightly decreased its content Conclusions Febuxostat significantly attenuated renal damage in STZ-induceddiabetic rats in addition to exerting hypouricemic effect

1 Introduction

Diabetic kidney disease (DKD) is the leading cause ofend stage renal failure (ESRD) worldwide [1] For manyyears several mechanisms including renal hemodynamicalterations renin-angiotensin-aldosterone system (RAAS)activation inflammatory pathways and reactive oxygenspecies (ROS) were widely studied in DKD and variouscorresponding therapeutic agents have been developed [2]However DKD outcomes following administration of thesetherapeutic agents offered no promising improvement [1]The underlying mechanisms and interventional targets ofDKD should be essentially explored

Several cohort and cross-sectional studies definitivelyestablished the relationship between hyperuricemia and theprogress of DKD in either type 1 or type 2 diabetes [3ndash5]Several clinical studies using hypouricemic agents such asallopurinol showed positive outcomes such as improvingrenal damage and postponing renal failure in patients witheither diabetes or chronic kidney disease (CKD) [6] Febux-ostat (Fx) is a recently developed xanthine oxidase (XO)inhibitor which has been definitively proved to be effectiveand safe for gout treatment [7] XO is an enzyme thatgenerates ROS by catalyzing the oxidation of hypoxanthineto xanthine and xanthine to uric acid Some of the renalprotective effects of Fx were clarified in animal models with

HindawiInternational Journal of NephrologyVolume 2017 Article ID 2739539 9 pageshttpsdoiorg10115520172739539

2 International Journal of Nephrology

One-week feeding with normal chow

Adaptive feeding for 2 weeks

Eight-week feeding with normal chow

Vital signs were measured and the blood and urine samples were taken at basal site justbefore Fx treatment and then repeated at the 4th and 8th week

At the 8th week all rats were sacrificed and livers were extracted finally

Diabetic ratswithout Fx (DM)(n = 12)

Diabetic rats with

(n = 12)Fx (DM + Fx)

Eight-week-old male Sprague-Dawley rats (n = 44)

Control ratswithout Fx (NC)(n = 10)

Control rats with

(n = 10)Fx (NC + Fx)

citrate buffer(n = 20)

Control withInduction ofdiabetes with

streptozotocin(n = 24)

Figure 1 Schematic diagram of the animal experiment protocol Fx febuxostat NC the normal control group without Fx treatment NC+ Fx the normal group with Fx treatment DM diabetic mellitus group without Fx treatment DM + Fx diabetic mellitus group with Fxtreatment

diabetes such as dbdb mice [8] and diabetic Zucker rats[9] Despite these promising data we noticed that the plasmauric acid (UA) levels in most studies were normal or evenlow because of the degradation of uricase an enzyme thatconverts uric acid to allantoin which is much more solublethan uric acid [10] Alterations in UA metabolism were alsoseldom discussed in these papers Suitable animal modelscharacterized by both diabetes and hyperuricemia should beexplored for researches in this field

In our previous studies [11] on streptozotocin- (STZ-)induced diabetic rats we found that serumUA concentrationwas significantly and permanently increased which wasaccompanied by abnormalities in renal function includingincreased serum creatinine (Scr) and albuminuria enlargedglomeruli and tubular hyalinization were also prominentin these rats Similar increase in UA concentration wasseen in STZ-induced diabetic rats in other studies [12 13]Therefore this rat model is more appropriate for researcheson hyperuricemia in diabetic conditions In the present studywe investigated the effects of Fx on renal injury in STZ-induced diabetic rats with the aim to search novel therapeuticmethod for DKD

2 Materials and Methods

21 Animal Preparation The overall animal experiment pro-tocol is shown in Figure 1 Eight-week-old male Sprague-Dawley rats (GuangdongMedical Laboratory Animal CenterFoshan China) weighing 200ndash220 g were adopted for thisstudy All rats were collectively housed (2 rats per cage)

and fed with standard rat chow for 2 weeks For diabetesinduction the rats were intraperitoneally injected with STZ(dissolved in 50mM citrate pH = 42 Sigma St Louis USA)at a single dose of 65mgkg Twenty-four rats with randomblood glucose levels gt 167mmolL at three different timeswere selected for the experiments Twenty rats that served ascontrolswere intraperitoneally injectedwith the same volumeof citrate buffer

22 Fx Treatment and Animal Experiments After successfulinduction of diabetes for 2 weeks the experimental diabeticand control ratswere treatedwith Fx (Melone PharmaceuticalCo Ltd Dalian China) which was dissolved in 05 car-boxymethylcellulose sodium (CMC-Na Fu Chen ChemicalReagents Factory Tianjin China) at a dose of 5mgkgd viadaily gavage for 8 weeks (Figure 1) The control rats weretreated only with the same volume of CMC-Na The ratswere divided into 4 groups during this intervention periodas follows diabetic rats with (DM + Fx 푛 = 12) and without(DM 푛 = 12) Fx treatment aswell as normal control ratswith(NC + Fx 푛 = 10) and without (NC 푛 = 10) Fx treatment

Vital signs including systolic blood pressure (SBP)diastolic blood pressure (DBP) and heart rate (HR) wererecorded in fully conscious rats by using indirect tail-cuffequipment (LE5002 Harvard Apparatus USA) After pre-warming the rats for 20min on a 37∘C plate the SBP DBPand HR of each rat were recorded

Blood and urine samples were collected every 4 weeksat the baseline and at weeks 4 and 8 respectively For urinesampling the rats were individually housed in metabolic

International Journal of Nephrology 3

cages for 24 h then all urine samples were collected andvolumetrically estimatedAll ratswere sacrificed after 8weeksof Fx treatment and the livers were removed for histologicenzymatic and genetic assays

During the whole experiment all rats were allowed freeaccess to standard rat chow and water and the room lightwas rotated at a 12-h light-dark cycle On the morning of theexperiment foods were withdrawn 12 h before each opera-tion All animal experimental procedures were approved bythe Ethnic Committee of Guangzhou Red Cross Hospital

23 Biochemical Assays The serum concentrations of glu-cose triglyceride (TG) total cholesterol (TC) uric acid(SUA) urea nitrogen (BUN) and SCr were measured usingthe corresponding commercial kits on an automatic bio-chemical machine (ECHO ECHO Italy) The 24-h urinesamples were collected and quantified Urinary uric acid(UUA) urinary urea nitrogen (UUN) and urinary creatinine(UCr)were detected by the same automaticmachine Urinaryalbumin was determined by the standard bromocresol greenmethod and the 24 h amount of urinary albumin excretion(UAE) was then calculated

24 Hepatic Content and Activity of XO The hepatic contentof XO was measured according to the previously describedmethod [14] Liver tissues weighing 025 g were mixed with9 times the volume of purified water and homogenizedThis mixture was centrifuged for 10min at 3000 rpm andthe supernatant was separated The XO concentration in thesupernatant was measured by using the corresponding com-mercial ELISA kits (Huamei Bioengineer Ltd Co WuhanChina)

For the measurement of hepatic XO activity we deter-mined the total protein content of the homogenate accord-ing to the Coomassie brilliant blue method [15] using acommercial reagent kit (Nanjing Jiancheng BioengineeringInstitute Nanjing China) Substrate and buffers were addedin the test and control reaction system (Nanjing JianchengBioengineering Institute Nanjing China) respectively Theabsorbance was measured at 530 nm after 20min incubationat 37∘C Hepatic XO activity was calculated according to theabsorbance difference and expressed as Ug protein

25 Gene Expression Hepatic gene expression of XO thekey enzyme for UA formation was determined by real-timepolymerase chain reaction (RT-PCR) Frozen tissues werehomogenized and total RNA was extracted using a TRIzolkit (Invitrogen CA USA) RNA quality and quantity wereassessed by automated capillary gel electrophoresis on aBioanalyzer 2100 with RNA Nano LabChips (Agilent Tech-nology Tokyo Japan) Then total RNA (1 휇g) was reverselytranscribed using a cDNA synthesis kit (Promega CA USA)with random primers in a 20휇L PCR system according to themanufacturerrsquos protocol Quantitative PCR was performedby SYBR Green PCR Master Mix (Toyobo Osaka Japan)and ABI PRISM 7500 Sequence Detection System (AppliedBiosystems Inc CA USA) Thermal cycling was carried outat 95∘C for 15min followed by 40 cycles at 95∘C for 15 s

60∘C for 15 s and 72∘C for 32 sWe used 18S rRNA as a house-keeping gene in RT-PCR The specific primers were selectedas follows XO forward 51015840-GACAGGGTGTTTATGAAGCA-31015840 XO reverse 51015840-AACTCACTGCGCTCGTATAG-31015840 18SrRNA forward 51015840-CCTGGATACCGCAGCTAGGA-31015840 18SrRNA reverse 51015840-GCGGCGCAATACGAATGCCCC-31015840

26 Statistical Analysis The results are expressed as mean plusmnSD One-way analysis of variance (ANOVA) and MannndashWhitney 푈 test were selected for comparisons of differ-ences between means and nonnormally distributed datadifferences respectively Statistical difference was accepted at푃 lt 005

3 Results

31 General Characteristics Although the diabetic rats weremore polyphagous and polydipsic than normal rats afterinduction of diabetes Fx treatment reduced the daily foodintake in diabetic rats at the 4th and 8th weeks (Figure 2both 푃 lt 005) The daily water intake was not affected byFx treatment in any rats Diabetic rats lost their weightsignificantly while the body weight of normal rats increasedcontinuously during the whole experiment In case of vitalsigns the diabetic rats showed lower HR than normal rats atthe 8th week (푃 lt 005) SBP and DBP decreased in diabeticrats at this time point (푃 lt 005) However Fx did not exertany effects on these vital signs either in diabetic or normalrats

32 Blood and Urine Biochemistry The blood biochemicalprofile throughout the experiment is shown in Figure 3After successful induction of diabetes the fasting plasmaglucose (FPG) in diabetic rats was maintained at very highlevels during the experiment compared to that in normalrats (all 푃 lt 005) NC + Fx rats showed slightly higherplasma TG than the NC control group (푃 lt 005) whilethe plasma TC level was comparable among all rat groupsIn both diabetic and normal rats Fx treatment did not alterthe plasma glucose and lipid levels SUA SCr and BUN levelsincreased significantly (all 푃 lt 005) in diabetic rats at thebaseline In diabetic rats Fx treatment slightly decreased SUAby 189 at the 8th week (푃 lt 005) while both SCr and BUNwere significantly decreased to around 300 (both푃 lt 005)SUA SCr and BUN were not affected by Fx treatment innormal rats (all 푃 gt 005)

Daily urinary excretions are depicted in Figure 4 UAE(at week 0 14942 plusmn 2985mgd 14004 plusmn 3011mgd 889 plusmn196mgd and 1052 plusmn 239mgd for DM DM + Fx NCand NC + Fx group resp) UUA UCr and UUN remarkablyincreased in the diabetic rats (all 푃 lt 005 when DMwas compared with NC group) Fx treatment significantlydecreased the daily UAE level at the 4th (12284plusmn 3265mgdfor DM and 9925 plusmn 3125mgd for DM + Fx 푃 lt 005)and 8th week (13821 plusmn 2257mgd for DM and 11084 plusmn2918mgd for DM + Fx 푃 lt 005) in diabetic rats Notablyin diabetic rats Fx treatment significantly increased UUA atthe 4th and 8th weeks (both 푃 lt 005) In particular at the

4 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0102030405060

Food

inta

ke (g

d) lowast lowastdagger lowastdagger

0

50

100

150

200

250

Wat

er in

take

(mld

)

0100200300400500600

Body

wei

ght (

g)

020406080

100120140160

SBP

(mm

Hg)

0

20

40

60

80

100

120

DBP

(mm

Hg)

0100200300400500600

HR

(bea

tsm

in)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast

lowastlowast lowast

lowast

lowast

lowast

Figure 2 Time course of food intake water intake body weight and main vital signs during the experiment after induction of diabetesHeart rate (HR) systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded by an indirect tail-cuff method The circlewith solid and dashed lines represents NC and NC + Fx groups respectively while the block symbol with solid and dashed lines shows dataof DM and DM + Fx groups respectively Data are expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and daggercorrespondingly when the DM + Fx group was compared with NC NC + Fx and DM groups

8th week the daily UUA was significantly increased to 520following Fx treatment UCr and UUN were also increasedafter Fx treatment at the 4th week (both 푃 lt 005) but theywere comparable at the 8th week (both 푃 gt 005) Fx didnot change the aforementioned daily urinary excretions innormal rats during the experiment

33 Hepatic Content Activity and Gene Expression of XOThe hepatic content of XO (12559 plusmn 304 ngmL for the DMgroup versus 5994 plusmn 323 ngmL for the CON group 푃 lt005) increased significantly in diabetic rats (Figure 5(a)) Inaddition the enzymatic activity of XO (Figure 5(b)) increasedsignificantly in diabetic rats compared to that in normalcontrol rats (2442plusmn 295Ug protein for theDMgroup versus1860 plusmn 216Ug protein for the CON group 푃 lt 005) Geneexpression of XO showed the same trend as that of hepaticcontent and enzymatic activity (Figure 5(c))

Fx treatment slightly decreased the hepatic XO contentin diabetic rats (Figure 5(a) 푃 lt 005) Nevertheless thetreatment exerted no effects on hepatic enzymatic activity

and gene expression of XO in either diabetic or normal rats(Figures 5(b) and 5(c) all 푃 gt 005)

4 Discussion

Similar to our previous study [11] we found that STZ-induced diabetic rats developed high levels of SUA and renaldamagemarked by elevated serumBUN SCr and dailyUAEFx a specific XO inhibitor significantly reduced SUA andattenuated renal functionwithout affecting the blood glucoseblood pressure and lipid profileThese findings indicated thathyperuricemia and its related pathological processes could bean important and directmechanismunderlying renal damagein STZ-induced diabetic rats Correspondingly all therapiesfocusing on UA metabolism may retard the progression ofdiabetic renal injury [16]

It remains unclear how UA directly facilitates renal dam-age in diabetic patients and various diabetic animal models[17] It is well known that RAAS triggers hemodynamicchanges and inflammatory attacks therefore it plays pivotal

International Journal of Nephrology 5

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

100

200

300

400FP

G (m

mol

L)

00020406081012

TG (m

mol

L)

0

20

40

60

80

100

120

SCr (

120583m

olL

)

0020406080

100120140160180

BUN

(mm

olL

)

00051015202530

TC (m

mol

L)

0100200300400500600700

SUA

(120583m

olL

)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast lowast

lowast lowast

lowastdagger lowastdagger

lowastdagger lowastdaggerlowastdagger

lowastdagger

lowast

Figure 3 Time course of blood biochemical indices among the four rat groups The circle with solid and dashed lines represents NC andNC + Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Dataare expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM + Fx group wascompared with NC NC + Fx and DM groups

roles in DKD [18] In vivo studies [19 20] have demon-strated that UA may promote RAAS activity in CKD animalmodels Several researches have reported links between UAmetabolism and other proinflammatory pathways [21 22]Among them the convincing one is that UA as crystals caus-ing cellular necrosis can activate the inflammasome NLRP3which consequently induces caspase-1 and its downstreamcytokines including IL-1훽 and IL-18 [23 24] The latter twocytokines have been proved to be potentially expressed ontubular epithelial cells and may closely relate to UA-inducedinterstitial damage [25]

The present study showed that Fx significantly reducedSUA by 18 and attenuated renal damage This result isconsistent with that of several other animal and clinicalstudies In diabetic dbdb mice [26] Kosugi et al foundthat tubulointerstitial injury is significantly attenuated bytreatmentwith allopurinol another XO inhibitor for 8weeksFollowing Fx treatment normalization of SUA and improve-ments in renal injury were also achieved in several diabeticmodels such as dbdb mice [8 27] Zucker diabetic rats [9]and STZ-induced diabetic rats [28] Large-scale clinical trials

on the effects of lowering SUA on DKD progression are stillscarce [29] In a previous study in type 2 diabetic patientswith DKD daily UAE was significantly reduced after a 4-month intervention with allopurinol [30] Recently anotherstudy [31] showed that 3-year treatment with allopurinol intype 2 diabetic patients with asymptomatic hyperuricemiadecreased UAE and SCr while the glomerular filtration ratewas increased In the present study daily UUN UCr andUUA increasing significantly after Fx treatment might beattributed to improved glomerular filtration we regarded thisas a novel finding if compared with other animal studies

Compared with the above-mentioned animal researchesthe most noticeable thing in our experiment is that Fxtreatment decreased SUA to approximately 18 in diabeticrats but in normal rats SUA was unchanged at the 8thweek Thus SUA in DM + Fx group was still signifi-cantly higher than that in both NC and NC + Fx groupsAccording to previous studies Fx at 5mgkg is a moderatedose for rats and mice [32] however we did not observecomplete normalization of SUA when compared to that infew other studies involving SUA [33] One reason for this

6 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

500

1000

1500

2000

UA

E (m

gd)

0005101520253035404550

UU

N (m

mol

d)

00102030405060708090

100

UCr

(120583m

old

)

00

50

100

150

200

250

UUA

(120583m

old

)NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowast

lowast

lowast

lowastlowastlowast

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Figure 4 Time course of daily urinary excretions among the four rat groups The circle with solid and dashed lines represents NC and NC+ Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Data areexpressed asmean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM+ Fx group was comparedwith NC NC + Fx and DM groups

CON DM0

20406080

100120140

XO co

nten

t (ng

mL)

CON + Fx DM + Fx

120576 lowastdagger

(a)CON DM

05

1015202530

XO ac

tivity

(Ug

pro

tein

)

CON + Fx DM + Fx

120576 lowast

(b)

NC DM000200400600800

1000120014001600

XO g

ene

DM + FxNC + Fx

120576 lowast

(c)

Figure 5Hepatic content activity and gene expression of xanthine oxidase (XO)Hepatic content ofXO (a)was expressed as its concentrationin the supernatant of liver extract Hepatic XO activity (b) was calculated according to the XO protein ratio and expressed as Ug proteinRelative gene expression (c) was determined by RT-PCR and shown in the graph as the amounts of initial template Data are expressed asmean plusmn SD 120576푃 lt 005 when the DM group was compared with CON or CON + Fx group lowast푃 lt 005 when DM + Fx group was comparedwith CON or CON + Fx and dagger when compared with DM group respectively

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

International Journal of Nephrology 3

cages for 24 h then all urine samples were collected andvolumetrically estimatedAll ratswere sacrificed after 8weeksof Fx treatment and the livers were removed for histologicenzymatic and genetic assays

During the whole experiment all rats were allowed freeaccess to standard rat chow and water and the room lightwas rotated at a 12-h light-dark cycle On the morning of theexperiment foods were withdrawn 12 h before each opera-tion All animal experimental procedures were approved bythe Ethnic Committee of Guangzhou Red Cross Hospital

23 Biochemical Assays The serum concentrations of glu-cose triglyceride (TG) total cholesterol (TC) uric acid(SUA) urea nitrogen (BUN) and SCr were measured usingthe corresponding commercial kits on an automatic bio-chemical machine (ECHO ECHO Italy) The 24-h urinesamples were collected and quantified Urinary uric acid(UUA) urinary urea nitrogen (UUN) and urinary creatinine(UCr)were detected by the same automaticmachine Urinaryalbumin was determined by the standard bromocresol greenmethod and the 24 h amount of urinary albumin excretion(UAE) was then calculated

24 Hepatic Content and Activity of XO The hepatic contentof XO was measured according to the previously describedmethod [14] Liver tissues weighing 025 g were mixed with9 times the volume of purified water and homogenizedThis mixture was centrifuged for 10min at 3000 rpm andthe supernatant was separated The XO concentration in thesupernatant was measured by using the corresponding com-mercial ELISA kits (Huamei Bioengineer Ltd Co WuhanChina)

For the measurement of hepatic XO activity we deter-mined the total protein content of the homogenate accord-ing to the Coomassie brilliant blue method [15] using acommercial reagent kit (Nanjing Jiancheng BioengineeringInstitute Nanjing China) Substrate and buffers were addedin the test and control reaction system (Nanjing JianchengBioengineering Institute Nanjing China) respectively Theabsorbance was measured at 530 nm after 20min incubationat 37∘C Hepatic XO activity was calculated according to theabsorbance difference and expressed as Ug protein

25 Gene Expression Hepatic gene expression of XO thekey enzyme for UA formation was determined by real-timepolymerase chain reaction (RT-PCR) Frozen tissues werehomogenized and total RNA was extracted using a TRIzolkit (Invitrogen CA USA) RNA quality and quantity wereassessed by automated capillary gel electrophoresis on aBioanalyzer 2100 with RNA Nano LabChips (Agilent Tech-nology Tokyo Japan) Then total RNA (1 휇g) was reverselytranscribed using a cDNA synthesis kit (Promega CA USA)with random primers in a 20휇L PCR system according to themanufacturerrsquos protocol Quantitative PCR was performedby SYBR Green PCR Master Mix (Toyobo Osaka Japan)and ABI PRISM 7500 Sequence Detection System (AppliedBiosystems Inc CA USA) Thermal cycling was carried outat 95∘C for 15min followed by 40 cycles at 95∘C for 15 s

60∘C for 15 s and 72∘C for 32 sWe used 18S rRNA as a house-keeping gene in RT-PCR The specific primers were selectedas follows XO forward 51015840-GACAGGGTGTTTATGAAGCA-31015840 XO reverse 51015840-AACTCACTGCGCTCGTATAG-31015840 18SrRNA forward 51015840-CCTGGATACCGCAGCTAGGA-31015840 18SrRNA reverse 51015840-GCGGCGCAATACGAATGCCCC-31015840

26 Statistical Analysis The results are expressed as mean plusmnSD One-way analysis of variance (ANOVA) and MannndashWhitney 푈 test were selected for comparisons of differ-ences between means and nonnormally distributed datadifferences respectively Statistical difference was accepted at푃 lt 005

3 Results

31 General Characteristics Although the diabetic rats weremore polyphagous and polydipsic than normal rats afterinduction of diabetes Fx treatment reduced the daily foodintake in diabetic rats at the 4th and 8th weeks (Figure 2both 푃 lt 005) The daily water intake was not affected byFx treatment in any rats Diabetic rats lost their weightsignificantly while the body weight of normal rats increasedcontinuously during the whole experiment In case of vitalsigns the diabetic rats showed lower HR than normal rats atthe 8th week (푃 lt 005) SBP and DBP decreased in diabeticrats at this time point (푃 lt 005) However Fx did not exertany effects on these vital signs either in diabetic or normalrats

32 Blood and Urine Biochemistry The blood biochemicalprofile throughout the experiment is shown in Figure 3After successful induction of diabetes the fasting plasmaglucose (FPG) in diabetic rats was maintained at very highlevels during the experiment compared to that in normalrats (all 푃 lt 005) NC + Fx rats showed slightly higherplasma TG than the NC control group (푃 lt 005) whilethe plasma TC level was comparable among all rat groupsIn both diabetic and normal rats Fx treatment did not alterthe plasma glucose and lipid levels SUA SCr and BUN levelsincreased significantly (all 푃 lt 005) in diabetic rats at thebaseline In diabetic rats Fx treatment slightly decreased SUAby 189 at the 8th week (푃 lt 005) while both SCr and BUNwere significantly decreased to around 300 (both푃 lt 005)SUA SCr and BUN were not affected by Fx treatment innormal rats (all 푃 gt 005)

Daily urinary excretions are depicted in Figure 4 UAE(at week 0 14942 plusmn 2985mgd 14004 plusmn 3011mgd 889 plusmn196mgd and 1052 plusmn 239mgd for DM DM + Fx NCand NC + Fx group resp) UUA UCr and UUN remarkablyincreased in the diabetic rats (all 푃 lt 005 when DMwas compared with NC group) Fx treatment significantlydecreased the daily UAE level at the 4th (12284plusmn 3265mgdfor DM and 9925 plusmn 3125mgd for DM + Fx 푃 lt 005)and 8th week (13821 plusmn 2257mgd for DM and 11084 plusmn2918mgd for DM + Fx 푃 lt 005) in diabetic rats Notablyin diabetic rats Fx treatment significantly increased UUA atthe 4th and 8th weeks (both 푃 lt 005) In particular at the

4 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0102030405060

Food

inta

ke (g

d) lowast lowastdagger lowastdagger

0

50

100

150

200

250

Wat

er in

take

(mld

)

0100200300400500600

Body

wei

ght (

g)

020406080

100120140160

SBP

(mm

Hg)

0

20

40

60

80

100

120

DBP

(mm

Hg)

0100200300400500600

HR

(bea

tsm

in)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast

lowastlowast lowast

lowast

lowast

lowast

Figure 2 Time course of food intake water intake body weight and main vital signs during the experiment after induction of diabetesHeart rate (HR) systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded by an indirect tail-cuff method The circlewith solid and dashed lines represents NC and NC + Fx groups respectively while the block symbol with solid and dashed lines shows dataof DM and DM + Fx groups respectively Data are expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and daggercorrespondingly when the DM + Fx group was compared with NC NC + Fx and DM groups

8th week the daily UUA was significantly increased to 520following Fx treatment UCr and UUN were also increasedafter Fx treatment at the 4th week (both 푃 lt 005) but theywere comparable at the 8th week (both 푃 gt 005) Fx didnot change the aforementioned daily urinary excretions innormal rats during the experiment

33 Hepatic Content Activity and Gene Expression of XOThe hepatic content of XO (12559 plusmn 304 ngmL for the DMgroup versus 5994 plusmn 323 ngmL for the CON group 푃 lt005) increased significantly in diabetic rats (Figure 5(a)) Inaddition the enzymatic activity of XO (Figure 5(b)) increasedsignificantly in diabetic rats compared to that in normalcontrol rats (2442plusmn 295Ug protein for theDMgroup versus1860 plusmn 216Ug protein for the CON group 푃 lt 005) Geneexpression of XO showed the same trend as that of hepaticcontent and enzymatic activity (Figure 5(c))

Fx treatment slightly decreased the hepatic XO contentin diabetic rats (Figure 5(a) 푃 lt 005) Nevertheless thetreatment exerted no effects on hepatic enzymatic activity

and gene expression of XO in either diabetic or normal rats(Figures 5(b) and 5(c) all 푃 gt 005)

4 Discussion

Similar to our previous study [11] we found that STZ-induced diabetic rats developed high levels of SUA and renaldamagemarked by elevated serumBUN SCr and dailyUAEFx a specific XO inhibitor significantly reduced SUA andattenuated renal functionwithout affecting the blood glucoseblood pressure and lipid profileThese findings indicated thathyperuricemia and its related pathological processes could bean important and directmechanismunderlying renal damagein STZ-induced diabetic rats Correspondingly all therapiesfocusing on UA metabolism may retard the progression ofdiabetic renal injury [16]

It remains unclear how UA directly facilitates renal dam-age in diabetic patients and various diabetic animal models[17] It is well known that RAAS triggers hemodynamicchanges and inflammatory attacks therefore it plays pivotal

International Journal of Nephrology 5

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

100

200

300

400FP

G (m

mol

L)

00020406081012

TG (m

mol

L)

0

20

40

60

80

100

120

SCr (

120583m

olL

)

0020406080

100120140160180

BUN

(mm

olL

)

00051015202530

TC (m

mol

L)

0100200300400500600700

SUA

(120583m

olL

)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast lowast

lowast lowast

lowastdagger lowastdagger

lowastdagger lowastdaggerlowastdagger

lowastdagger

lowast

Figure 3 Time course of blood biochemical indices among the four rat groups The circle with solid and dashed lines represents NC andNC + Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Dataare expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM + Fx group wascompared with NC NC + Fx and DM groups

roles in DKD [18] In vivo studies [19 20] have demon-strated that UA may promote RAAS activity in CKD animalmodels Several researches have reported links between UAmetabolism and other proinflammatory pathways [21 22]Among them the convincing one is that UA as crystals caus-ing cellular necrosis can activate the inflammasome NLRP3which consequently induces caspase-1 and its downstreamcytokines including IL-1훽 and IL-18 [23 24] The latter twocytokines have been proved to be potentially expressed ontubular epithelial cells and may closely relate to UA-inducedinterstitial damage [25]

The present study showed that Fx significantly reducedSUA by 18 and attenuated renal damage This result isconsistent with that of several other animal and clinicalstudies In diabetic dbdb mice [26] Kosugi et al foundthat tubulointerstitial injury is significantly attenuated bytreatmentwith allopurinol another XO inhibitor for 8weeksFollowing Fx treatment normalization of SUA and improve-ments in renal injury were also achieved in several diabeticmodels such as dbdb mice [8 27] Zucker diabetic rats [9]and STZ-induced diabetic rats [28] Large-scale clinical trials

on the effects of lowering SUA on DKD progression are stillscarce [29] In a previous study in type 2 diabetic patientswith DKD daily UAE was significantly reduced after a 4-month intervention with allopurinol [30] Recently anotherstudy [31] showed that 3-year treatment with allopurinol intype 2 diabetic patients with asymptomatic hyperuricemiadecreased UAE and SCr while the glomerular filtration ratewas increased In the present study daily UUN UCr andUUA increasing significantly after Fx treatment might beattributed to improved glomerular filtration we regarded thisas a novel finding if compared with other animal studies

Compared with the above-mentioned animal researchesthe most noticeable thing in our experiment is that Fxtreatment decreased SUA to approximately 18 in diabeticrats but in normal rats SUA was unchanged at the 8thweek Thus SUA in DM + Fx group was still signifi-cantly higher than that in both NC and NC + Fx groupsAccording to previous studies Fx at 5mgkg is a moderatedose for rats and mice [32] however we did not observecomplete normalization of SUA when compared to that infew other studies involving SUA [33] One reason for this

6 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

500

1000

1500

2000

UA

E (m

gd)

0005101520253035404550

UU

N (m

mol

d)

00102030405060708090

100

UCr

(120583m

old

)

00

50

100

150

200

250

UUA

(120583m

old

)NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowast

lowast

lowast

lowastlowastlowast

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Figure 4 Time course of daily urinary excretions among the four rat groups The circle with solid and dashed lines represents NC and NC+ Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Data areexpressed asmean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM+ Fx group was comparedwith NC NC + Fx and DM groups

CON DM0

20406080

100120140

XO co

nten

t (ng

mL)

CON + Fx DM + Fx

120576 lowastdagger

(a)CON DM

05

1015202530

XO ac

tivity

(Ug

pro

tein

)

CON + Fx DM + Fx

120576 lowast

(b)

NC DM000200400600800

1000120014001600

XO g

ene

DM + FxNC + Fx

120576 lowast

(c)

Figure 5Hepatic content activity and gene expression of xanthine oxidase (XO)Hepatic content ofXO (a)was expressed as its concentrationin the supernatant of liver extract Hepatic XO activity (b) was calculated according to the XO protein ratio and expressed as Ug proteinRelative gene expression (c) was determined by RT-PCR and shown in the graph as the amounts of initial template Data are expressed asmean plusmn SD 120576푃 lt 005 when the DM group was compared with CON or CON + Fx group lowast푃 lt 005 when DM + Fx group was comparedwith CON or CON + Fx and dagger when compared with DM group respectively

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0102030405060

Food

inta

ke (g

d) lowast lowastdagger lowastdagger

0

50

100

150

200

250

Wat

er in

take

(mld

)

0100200300400500600

Body

wei

ght (

g)

020406080

100120140160

SBP

(mm

Hg)

0

20

40

60

80

100

120

DBP

(mm

Hg)

0100200300400500600

HR

(bea

tsm

in)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast

lowastlowast lowast

lowast

lowast

lowast

Figure 2 Time course of food intake water intake body weight and main vital signs during the experiment after induction of diabetesHeart rate (HR) systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded by an indirect tail-cuff method The circlewith solid and dashed lines represents NC and NC + Fx groups respectively while the block symbol with solid and dashed lines shows dataof DM and DM + Fx groups respectively Data are expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and daggercorrespondingly when the DM + Fx group was compared with NC NC + Fx and DM groups

8th week the daily UUA was significantly increased to 520following Fx treatment UCr and UUN were also increasedafter Fx treatment at the 4th week (both 푃 lt 005) but theywere comparable at the 8th week (both 푃 gt 005) Fx didnot change the aforementioned daily urinary excretions innormal rats during the experiment

33 Hepatic Content Activity and Gene Expression of XOThe hepatic content of XO (12559 plusmn 304 ngmL for the DMgroup versus 5994 plusmn 323 ngmL for the CON group 푃 lt005) increased significantly in diabetic rats (Figure 5(a)) Inaddition the enzymatic activity of XO (Figure 5(b)) increasedsignificantly in diabetic rats compared to that in normalcontrol rats (2442plusmn 295Ug protein for theDMgroup versus1860 plusmn 216Ug protein for the CON group 푃 lt 005) Geneexpression of XO showed the same trend as that of hepaticcontent and enzymatic activity (Figure 5(c))

Fx treatment slightly decreased the hepatic XO contentin diabetic rats (Figure 5(a) 푃 lt 005) Nevertheless thetreatment exerted no effects on hepatic enzymatic activity

and gene expression of XO in either diabetic or normal rats(Figures 5(b) and 5(c) all 푃 gt 005)

4 Discussion

Similar to our previous study [11] we found that STZ-induced diabetic rats developed high levels of SUA and renaldamagemarked by elevated serumBUN SCr and dailyUAEFx a specific XO inhibitor significantly reduced SUA andattenuated renal functionwithout affecting the blood glucoseblood pressure and lipid profileThese findings indicated thathyperuricemia and its related pathological processes could bean important and directmechanismunderlying renal damagein STZ-induced diabetic rats Correspondingly all therapiesfocusing on UA metabolism may retard the progression ofdiabetic renal injury [16]

It remains unclear how UA directly facilitates renal dam-age in diabetic patients and various diabetic animal models[17] It is well known that RAAS triggers hemodynamicchanges and inflammatory attacks therefore it plays pivotal

International Journal of Nephrology 5

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

100

200

300

400FP

G (m

mol

L)

00020406081012

TG (m

mol

L)

0

20

40

60

80

100

120

SCr (

120583m

olL

)

0020406080

100120140160180

BUN

(mm

olL

)

00051015202530

TC (m

mol

L)

0100200300400500600700

SUA

(120583m

olL

)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast lowast

lowast lowast

lowastdagger lowastdagger

lowastdagger lowastdaggerlowastdagger

lowastdagger

lowast

Figure 3 Time course of blood biochemical indices among the four rat groups The circle with solid and dashed lines represents NC andNC + Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Dataare expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM + Fx group wascompared with NC NC + Fx and DM groups

roles in DKD [18] In vivo studies [19 20] have demon-strated that UA may promote RAAS activity in CKD animalmodels Several researches have reported links between UAmetabolism and other proinflammatory pathways [21 22]Among them the convincing one is that UA as crystals caus-ing cellular necrosis can activate the inflammasome NLRP3which consequently induces caspase-1 and its downstreamcytokines including IL-1훽 and IL-18 [23 24] The latter twocytokines have been proved to be potentially expressed ontubular epithelial cells and may closely relate to UA-inducedinterstitial damage [25]

The present study showed that Fx significantly reducedSUA by 18 and attenuated renal damage This result isconsistent with that of several other animal and clinicalstudies In diabetic dbdb mice [26] Kosugi et al foundthat tubulointerstitial injury is significantly attenuated bytreatmentwith allopurinol another XO inhibitor for 8weeksFollowing Fx treatment normalization of SUA and improve-ments in renal injury were also achieved in several diabeticmodels such as dbdb mice [8 27] Zucker diabetic rats [9]and STZ-induced diabetic rats [28] Large-scale clinical trials

on the effects of lowering SUA on DKD progression are stillscarce [29] In a previous study in type 2 diabetic patientswith DKD daily UAE was significantly reduced after a 4-month intervention with allopurinol [30] Recently anotherstudy [31] showed that 3-year treatment with allopurinol intype 2 diabetic patients with asymptomatic hyperuricemiadecreased UAE and SCr while the glomerular filtration ratewas increased In the present study daily UUN UCr andUUA increasing significantly after Fx treatment might beattributed to improved glomerular filtration we regarded thisas a novel finding if compared with other animal studies

Compared with the above-mentioned animal researchesthe most noticeable thing in our experiment is that Fxtreatment decreased SUA to approximately 18 in diabeticrats but in normal rats SUA was unchanged at the 8thweek Thus SUA in DM + Fx group was still signifi-cantly higher than that in both NC and NC + Fx groupsAccording to previous studies Fx at 5mgkg is a moderatedose for rats and mice [32] however we did not observecomplete normalization of SUA when compared to that infew other studies involving SUA [33] One reason for this

6 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

500

1000

1500

2000

UA

E (m

gd)

0005101520253035404550

UU

N (m

mol

d)

00102030405060708090

100

UCr

(120583m

old

)

00

50

100

150

200

250

UUA

(120583m

old

)NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowast

lowast

lowast

lowastlowastlowast

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Figure 4 Time course of daily urinary excretions among the four rat groups The circle with solid and dashed lines represents NC and NC+ Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Data areexpressed asmean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM+ Fx group was comparedwith NC NC + Fx and DM groups

CON DM0

20406080

100120140

XO co

nten

t (ng

mL)

CON + Fx DM + Fx

120576 lowastdagger

(a)CON DM

05

1015202530

XO ac

tivity

(Ug

pro

tein

)

CON + Fx DM + Fx

120576 lowast

(b)

NC DM000200400600800

1000120014001600

XO g

ene

DM + FxNC + Fx

120576 lowast

(c)

Figure 5Hepatic content activity and gene expression of xanthine oxidase (XO)Hepatic content ofXO (a)was expressed as its concentrationin the supernatant of liver extract Hepatic XO activity (b) was calculated according to the XO protein ratio and expressed as Ug proteinRelative gene expression (c) was determined by RT-PCR and shown in the graph as the amounts of initial template Data are expressed asmean plusmn SD 120576푃 lt 005 when the DM group was compared with CON or CON + Fx group lowast푃 lt 005 when DM + Fx group was comparedwith CON or CON + Fx and dagger when compared with DM group respectively

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

International Journal of Nephrology 5

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

100

200

300

400FP

G (m

mol

L)

00020406081012

TG (m

mol

L)

0

20

40

60

80

100

120

SCr (

120583m

olL

)

0020406080

100120140160180

BUN

(mm

olL

)

00051015202530

TC (m

mol

L)

0100200300400500600700

SUA

(120583m

olL

)

NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowastlowast

lowast lowast

lowast lowast

lowastdagger lowastdagger

lowastdagger lowastdaggerlowastdagger

lowastdagger

lowast

Figure 3 Time course of blood biochemical indices among the four rat groups The circle with solid and dashed lines represents NC andNC + Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Dataare expressed as mean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM + Fx group wascompared with NC NC + Fx and DM groups

roles in DKD [18] In vivo studies [19 20] have demon-strated that UA may promote RAAS activity in CKD animalmodels Several researches have reported links between UAmetabolism and other proinflammatory pathways [21 22]Among them the convincing one is that UA as crystals caus-ing cellular necrosis can activate the inflammasome NLRP3which consequently induces caspase-1 and its downstreamcytokines including IL-1훽 and IL-18 [23 24] The latter twocytokines have been proved to be potentially expressed ontubular epithelial cells and may closely relate to UA-inducedinterstitial damage [25]

The present study showed that Fx significantly reducedSUA by 18 and attenuated renal damage This result isconsistent with that of several other animal and clinicalstudies In diabetic dbdb mice [26] Kosugi et al foundthat tubulointerstitial injury is significantly attenuated bytreatmentwith allopurinol another XO inhibitor for 8weeksFollowing Fx treatment normalization of SUA and improve-ments in renal injury were also achieved in several diabeticmodels such as dbdb mice [8 27] Zucker diabetic rats [9]and STZ-induced diabetic rats [28] Large-scale clinical trials

on the effects of lowering SUA on DKD progression are stillscarce [29] In a previous study in type 2 diabetic patientswith DKD daily UAE was significantly reduced after a 4-month intervention with allopurinol [30] Recently anotherstudy [31] showed that 3-year treatment with allopurinol intype 2 diabetic patients with asymptomatic hyperuricemiadecreased UAE and SCr while the glomerular filtration ratewas increased In the present study daily UUN UCr andUUA increasing significantly after Fx treatment might beattributed to improved glomerular filtration we regarded thisas a novel finding if compared with other animal studies

Compared with the above-mentioned animal researchesthe most noticeable thing in our experiment is that Fxtreatment decreased SUA to approximately 18 in diabeticrats but in normal rats SUA was unchanged at the 8thweek Thus SUA in DM + Fx group was still signifi-cantly higher than that in both NC and NC + Fx groupsAccording to previous studies Fx at 5mgkg is a moderatedose for rats and mice [32] however we did not observecomplete normalization of SUA when compared to that infew other studies involving SUA [33] One reason for this

6 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

500

1000

1500

2000

UA

E (m

gd)

0005101520253035404550

UU

N (m

mol

d)

00102030405060708090

100

UCr

(120583m

old

)

00

50

100

150

200

250

UUA

(120583m

old

)NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowast

lowast

lowast

lowastlowastlowast

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Figure 4 Time course of daily urinary excretions among the four rat groups The circle with solid and dashed lines represents NC and NC+ Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Data areexpressed asmean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM+ Fx group was comparedwith NC NC + Fx and DM groups

CON DM0

20406080

100120140

XO co

nten

t (ng

mL)

CON + Fx DM + Fx

120576 lowastdagger

(a)CON DM

05

1015202530

XO ac

tivity

(Ug

pro

tein

)

CON + Fx DM + Fx

120576 lowast

(b)

NC DM000200400600800

1000120014001600

XO g

ene

DM + FxNC + Fx

120576 lowast

(c)

Figure 5Hepatic content activity and gene expression of xanthine oxidase (XO)Hepatic content ofXO (a)was expressed as its concentrationin the supernatant of liver extract Hepatic XO activity (b) was calculated according to the XO protein ratio and expressed as Ug proteinRelative gene expression (c) was determined by RT-PCR and shown in the graph as the amounts of initial template Data are expressed asmean plusmn SD 120576푃 lt 005 when the DM group was compared with CON or CON + Fx group lowast푃 lt 005 when DM + Fx group was comparedwith CON or CON + Fx and dagger when compared with DM group respectively

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 International Journal of Nephrology

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

0 4 8(Week)

00

500

1000

1500

2000

UA

E (m

gd)

0005101520253035404550

UU

N (m

mol

d)

00102030405060708090

100

UCr

(120583m

old

)

00

50

100

150

200

250

UUA

(120583m

old

)NCNC + Fx

DMDM + Fx

NCNC + Fx

DMDM + Fx

lowast

lowast

lowast

lowastlowastlowast

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Figure 4 Time course of daily urinary excretions among the four rat groups The circle with solid and dashed lines represents NC and NC+ Fx groups respectively while the block symbol with solid and dashed lines shows data of DM and DM + Fx groups respectively Data areexpressed asmean plusmn SD Statistical significance (푃 lt 005) was labeled as lowast and dagger correspondingly when the DM+ Fx group was comparedwith NC NC + Fx and DM groups

CON DM0

20406080

100120140

XO co

nten

t (ng

mL)

CON + Fx DM + Fx

120576 lowastdagger

(a)CON DM

05

1015202530

XO ac

tivity

(Ug

pro

tein

)

CON + Fx DM + Fx

120576 lowast

(b)

NC DM000200400600800

1000120014001600

XO g

ene

DM + FxNC + Fx

120576 lowast

(c)

Figure 5Hepatic content activity and gene expression of xanthine oxidase (XO)Hepatic content ofXO (a)was expressed as its concentrationin the supernatant of liver extract Hepatic XO activity (b) was calculated according to the XO protein ratio and expressed as Ug proteinRelative gene expression (c) was determined by RT-PCR and shown in the graph as the amounts of initial template Data are expressed asmean plusmn SD 120576푃 lt 005 when the DM group was compared with CON or CON + Fx group lowast푃 lt 005 when DM + Fx group was comparedwith CON or CON + Fx and dagger when compared with DM group respectively

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

International Journal of Nephrology 7

might result from the relatively short-term intervention ofFx (only 8 weeks) Second we speculate that STZ mightaggravate UA metabolism by directly suppressing uricaseto some extent instead of promoting in vivo XO activity[34] Significantly increased hepatic XO gene expressioncontent and activity in diabetic rats were observed in ourresearch which were similar to those observed in severalstudies using other diabetic models such as Zucker diabeticrats [9] Otsuka Long-Evans Tokushima fatty rats [35] anddbdb mice [8] We may conclude that diabetes itself ratherthan STZ activates XO thereby promoting UA productionin these rats [36] This hypothesis can illustrate why Fx onlyslightly decreases SUA in STZ-induced diabetic rats whenno hypoglycemic treatment is provided Further studies areneeded to elucidate the relationship between STZ and UAmetabolism

Despite the slight reduction of SUA remarkable renalprotective effects were observed in the diabetic rats DailyUAE significantly decreased and more importantly serumBUN and SCr decreased by more than 30 in these ratsFx treatment should specifically target XO and this caninhibit in vivo UA production [9] Hepatic content activityand gene expression were measured in our experiment Allthese parameters were significantly increased in diabeticrats in agreement with the findings of other studies [89 35] Nevertheless exceptional results were observed inthis study including unchanged XO gene expression andactivity and a slight decrease in hepatic XO content after Fxtreatment in diabetic rats Gene expression and activity ofXO were unchanged which may result from the upregulatingeffects after the competitive inhibition by Fx But anywayFx decreased SUA slightly and exerted minor effects onhepatic XO content Simultaneously Fx treatment did notmake significant changes in body weight blood pressureand metabolic indices including blood glucose and lipidlevels These data throw light on mechanisms other thanhypouricemic effect that may underlie the renal protectiveeffects of Fx

Several other researches explored the mechanisms in-volved in the attenuation of renal injury by Fx treatmentSanchez-Lozada et al found that Fx significantly reducedglomerular pressure and renal vasoconstriction in fructose-induced metabolic syndrome [33] and oxonic acid-inducedhyperuricemic rat models [37] Moreover in STZ-induceddiabetic rat models Lee et al [28] showed that Fx preventsrenal damage mainly by ameliorating the inflammatoryfactors and oxidative stress owing to its inhibitory effectson XO In a recent study in Zucker diabetic rats Komerset al [9] examined the effect of Fx on oxidative stressThe simultaneous inhibition of profibrotic signaling by Fxmight be another pivotal mechanism We did not performfurther experiments on glomerular structure and interstitialchanges in our study However it is well known [38] thatUA preferentially induces tubular damage In our previousstudy feeding the same diabetic rats with low protein dietsdecreased SUA and attenuated tubular injuries In the presentstudy we noticed that daily UAE was increased after Fxtreatment in diabetic rats which could not be appropriatelyexplained by its direct pharmaceutical actions [39] Unknown

but important mechanisms of Fx on renal tubular damageshould be investigated in future researches

This study has several limitations First the morphologicalterations of glomerular and interstitial area after Fx treat-ment should be studied in a long-term experiment Secondthe renal hemodynamic parameters were not included in thestudy design and these alterations may partly be responsiblefor the improvement of renal function in diabetic ratsResearches on the direct effects of UA on renal damage indiabetes have been initiated andmore compromised data willbe provided in the future

5 Conclusions

Fx attenuated renal damage without any significant influenceon glucose blood pressure and lipid levels in STZ-induceddiabetic and hyperuricemic rats The mild hypouricemiceffects and actions of Fx on XO pave the way for researchersto explore other underlying mechanisms especially in thetubules besides its traditional targets

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors greatly thank all the staffs of GuangdongMedicalLaboratory Animal Center for their excellent technical assis-tance in animalmanagement and experimentsThe study wassupported by Guangzhou Science and Technology ProjectFund (no 201300000181 no 2014Y2-00145 no 2014Y2-00166 no 14A33151295 and no 2014J4100076) GuangzhouScientific Fund for Clinical Study and TranslationalMedicine(no 2014Y2-00549) and Guangdong Science and Technol-ogy Project Fund for Key Scientific Research Base (no2014B030303002)

References

[1] M Afkarian L R Zelnick Y N Hall et al ldquoClinical man-ifestations of kidney disease among US adults with diabetes1988ndash2014rdquo The Journal of the American Medical Associationvol 316 no 6 pp 602ndash610 2016

[2] N Helou A Dwyer M Shaha and A Zanchi ldquoMultidisci-plinary management of diabetic kidney disease a systematicreview and meta-analysisrdquo JBI Database of Systematic Reviewsand Implementation Reports vol 14 pp 169ndash207 2016

[3] P Hovind P Rossing L Tarnow R J Johnson and H HParving ldquoSerum uric acid as a predictor for development ofdiabetic nephropathy in type 1 diabetes an inception cohortstudyrdquo Diabetes vol 58 pp 1668ndash1671 2009

[4] Y Hayashino S Okamura S Tsujii and H Ishii ldquoAssociationof serum uric acid levels with the risk of development orprogression of albuminuria among Japanese patients with type2 diabetes a prospective cohort study [Diabetes Distress andCare Registry at Tenri (DDCRT 10)]rdquo Acta Diabetologica vol53 no 4 pp 599ndash607 2016

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 International Journal of Nephrology

[5] Y-H Chang C-C Lei K-C Lin D-M Chang C-H Hsiehand Y-J Lee ldquoSerum uric acid level as an indicator forCKD regression and progression in patients with type 2 dia-betes mellitusmdasha 46-year cohort studyrdquo DiabetesMetabolismResearch and Reviews vol 32 pp 557ndash564 2016

[6] C Mende ldquoManagement of chronic kidney disease therelationship between serum uric acid and development ofnephropathyrdquoAdvances inTherapy vol 32 no 12 pp 1177ndash11912015

[7] S Li H Yang Y Guo et al ldquoComparative efficacy and safetyof urate-lowering therapy for the treatment of hyperuricemiaa systematic review and network meta-analysisrdquo ScientificReports vol 6 no 1 Article ID 33082 2016

[8] T Nakamura T Murase M Nampei et al ldquoEffects of topiroxo-stat and febuxostat on urinary albumin excretion and plasmaxanthine oxidoreductase activity in dbdb micerdquo EuropeanJournal of Pharmacology vol 780 pp 224ndash231 2016

[9] R Komers B Xu J Schneider and T T Oyama ldquoEffects ofxanthine oxidase inhibitionwith febuxostat on the developmentof nephropathy in experimental type 2 diabetesrdquo British Journalof Pharmacology vol 173 pp 2573ndash2588 2016

[10] J T Kratzer M A Lanaspa M N Murphy et al ldquoEvolutionaryhistory and metabolic insights of ancient mammalian uricasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 10 pp 3763ndash3768 2014

[11] J Ran J Ma Y Liu R Tan H Liu and G Lao ldquoLow proteindiet inhibits uric acid synthesis and attenuates renal damagein streptozotocin-induced diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 287536 2014

[12] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[13] D S Ibrahim and M A E Abd El-Maksoud ldquoEffect of straw-berry (Fragaria times ananassa) leaf extract on diabetic nephropa-thy in ratsrdquo International Journal of Experimental Pathology vol96 no 2 pp 87ndash93 2015

[14] L P Peters and R W Teel ldquoEffect of high sucrose diet onliver enzyme content and activity and aflatoxin B1-inducedmutagenesisrdquo In Vivo vol 17 no 2 pp 205ndash210 2003

[15] Y Jin and T Manabe ldquoHigh-efficiency protein extractionfrom polyacrylamide gels for molecular mass measurementby matrix-assisted laser desorptionionization-time of flight-mass spectrometryrdquoElectrophoresis vol 26 no 6 pp 1019ndash10282005

[16] P Hovind P Rossing R J Johnson andH-H Parving ldquoSerumuric acid as a new player in the development of diabeticnephropathyrdquo Journal of Renal Nutrition vol 21 no 1 pp 124ndash127 2011

[17] P Dousdampanis K Trigka C G Musso and C FourtounasldquoHyperuricemia and chronic kidney disease an enigma yet tobe solvedrdquo Renal Failure vol 36 no 9 pp 1351ndash1359 2014

[18] S S Roscioni H J L Heerspink and D De Zeeuw ldquoThe effectof RAAS blockade on the progression of diabetic nephropathyrdquoNature Reviews Nephrology vol 10 no 2 pp 77ndash87 2014

[19] A Eraranta V Kurra A M Tahvanainen et al ldquoOxonicacid-induced hyperuricemia elevates plasma aldosterone inexperimental renal insufficiencyrdquo Journal of Hypertension vol26 no 8 pp 1661ndash1668 2008

[20] D B Corry P Eslami K Yamamoto M D Nyby H Makinoand M L Tuck ldquoUric acid stimulates vascular smooth muscle

cell proliferation and oxidative stress via the vascular renin-angiotensin systemrdquo Journal of Hypertension vol 26 no 2 pp269ndash275 2008

[21] V Filiopoulos D Hadjiyannakos and D Vlassopoulos ldquoNewinsights into uric acid effects on the progression and prognosisof chronic kidney diseaserdquo Renal Failure vol 34 no 4 pp 510ndash520 2012

[22] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[23] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 pp 251ndash264 2016

[24] T T Braga M F Forni M Correa-Costa et al ldquoSoluble uricacid activates theNLRP3 inflammasomerdquo Scientific Reports vol7 article 39884 2017

[25] S-M Kim S-H Lee Y-G Kim et al ldquoHyperuricemia-induced NLRP3 activation of macrophages contributes to theprogression of diabetic nephropathyrdquo American Journal ofPhysiologymdashRenal Physiology vol 308 no 9 pp F993ndashF10032015

[26] T Kosugi T Nakayama M Heinig et al ldquoEffect of loweringuric acid on renal disease in the type 2 diabetic dbdb micerdquoAmerican Journal of PhysiologymdashRenal Physiology vol 297 no2 pp F481ndashF488 2009

[27] A Kushiyama K Tanaka S Hara and S Kawazu ldquoLinking uricacid metabolism to diabetic complicationsrdquo World Journal ofDiabetes vol 5 no 6 pp 787ndash795 2014

[28] H-J Lee K H Jeong Y G Kim et al ldquoFebuxostat amelioratesdiabetic renal injury in a streptozotocin-induced diabetic ratmodelrdquo American Journal of Nephrology vol 40 no 1 pp 56ndash63 2014

[29] D M Maahs L Caramori D Z I Cherney et al ldquoUricacid lowering to prevent kidney function loss in diabetes thepreventing early renal function loss (PERL) allopurinol studyrdquoCurrent Diabetes Reports vol 13 no 4 pp 550ndash559 2013

[30] A Momeni S Shahidi S Seirafian S Taheri and S KheirildquoEffect of allopurinol in decreasing proteinuria in type 2diabetic patientsrdquo Iranian Journal of Kidney Diseases vol 4 no2 pp 128ndash132 2010

[31] P Liu Y Chen B Wang F Zhang D Wang and Y WangldquoAllopurinol treatment improves renal function in patientswith type 2 diabetes and asymptomatic hyperuricemia 3-yearrandomized parallel-controlled studyrdquo Clinical Endocrinologyvol 83 no 4 pp 475ndash482 2015

[32] B Krishnamurthy N Rani S Bharti et al ldquoFebuxostat ame-liorates doxorubicin-induced cardiotoxicity in ratsrdquo Chemico-Biological Interactions vol 237 pp 96ndash103 2015

[33] L G Sanchez-Lozada E Tapia P Bautista-Garcıa et al ldquoEffectsof febuxostat on metabolic and renal alterations in rats withfructose-induced metabolic syndromerdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 4 pp F710ndashF7182008

[34] H Osmundsen B Brodal and R Hovik ldquoA luminomet-ric assay for peroxisomal 훽-oxidation Effects of fasting andstreptozotocin-diabetes on peroxisomal 훽-oxidationrdquo Biochem-ical Journal vol 260 no 1 pp 215ndash220 1989

[35] I J Kim Y K Kim S M Son K W Hong and C D KimldquoEnhanced vascular production of superoxide in OLETF ratafter the onset of hyperglycemiardquoDiabetes Research and ClinicalPractice vol 60 no 1 pp 11ndash18 2003

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

International Journal of Nephrology 9

[36] M Banerjee and P Vats ldquoReactive metabolites and antioxidantgene polymorphisms in Type 2 diabetes mellitusrdquo Redox Biol-ogy vol 2 no 1 pp 170ndash177 2014

[37] L G Sanchez-Lozada E Tapia V Soto et al ldquoTreatment withthe xanthine oxidase inhibitor febuxostat lowers uric acid andalleviates systemic and glomerular hypertension in experimen-tal hyperuricaemiardquo Nephrology Dialysis Transplantation vol23 no 4 pp 1179ndash1185 2008

[38] S A Fathallah-Shaykh and M T Cramer ldquoUric acid and thekidneyrdquo Pediatric Nephrology vol 29 no 6 pp 999ndash1008 2014

[39] B Grabowski R Khosravan J-T Wu L Vernillet and CLademacher ldquoEffect of hydrochlorothiazide on the pharma-cokinetics and pharmacodynamics of febuxostat a non-purineselective inhibitor of xanthine oxidaserdquo British Journal of Clini-cal Pharmacology vol 70 no 1 pp 57ndash64 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom