podocyte injury and albuminuria in experimental hyperuricemic...

Research ArticlePodocyte Injury and Albuminuria in ExperimentalHyperuricemic Model Rats

Shinichiro Asakawa1 Shigeru Shibata1 Chikayuki Morimoto1

Takeshi Shiraishi12 Takashi Nakamura3 Yoshifuru Tamura1

Takanori Kumagai12 Makoto Hosoyamada4 and Shunya Uchida1

1Division of Nephrology Department of Internal Medicine Teikyo University School of Medicine 2-11-1 KagaItabashi-ku Tokyo 173-8605 Japan2Support for Community Medicine Endowed Chair Teikyo University School of Medicine Tokyo 173-8605 Japan3Pharmacological Study Group Pharmaceutical Research Laboratories Sanwa Kagaku Kenkyusho Mie 511-0406 Japan4Department of Human Physiology and Pathology Faculty of Pharma-Sciences Teikyo University 2-11-1 KagaItabashi-ku Tokyo 173-8605 Japan

Correspondence should be addressed to Shigeru Shibata shigerushibatamedteikyo-uacjpand Shunya Uchida s uchidanetjoynejp

Received 22 October 2016 Revised 20 January 2017 Accepted 29 January 2017 Published 28 February 2017

Academic Editor Jose L Quiles

Copyright copy 2017 Shinichiro Asakawa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Although hyperuricemia is shown to accelerate chronic kidney disease the mechanisms remain unclear Accumulating studiesalso indicate that uric acid has both pro- and antioxidant properties We postulated that hyperuricemia impairs the function ofglomerular podocytes resulting in albuminuria Hyperuricemic model was induced by oral administration of 2 oxonic acid auricase inhibitor Oxonic acid caused a twofold increase in serum uric acid levels at 8 weeks when compared to control animalsHyperuricemia in this model was associated with the increase in blood pressure and the wall-thickening of afferent arterioles aswell as arcuate arteries Notably hyperuricemic rats showed significant albuminuria and the podocyte injury marker desmin wasupregulated in the glomeruli Conversely podocin the key component of podocyte slit diaphragm was downregulated Structuralanalysis using transmission electron microscopy confirmed podocyte injury in this model We found that urinary 8-hydroxy-21015840-deoxyguanosine levels were significantly increased and correlated with albuminuria and podocytopathy Interestingly althoughthe superoxide dismutase mimetic tempol ameliorated the vascular changes and the hypertension it failed to reduce albuminuriasuggesting that vascular remodeling and podocyte injury in this model are mediated through different mechanisms In conclusionvasculopathy and podocytopathy may distinctly contribute to the kidney injury in a hyperuricemic state

1 Introduction

Chronic kidney disease (CKD) continues to be a public healthproblem worldwide [1] CKD not only causes end-stagerenal disease (ESRD) but also increases the prevalence ofcardiovascular disease [2 3] and therefore early interventionagainst the risk factors for CKD is crucial to improve renaland cardiovascular outcomes Hyperuricemia has long beenspeculated as a possible risk factor of the incidence and pro-gression of CKD over the last decade but without reaching

a broad consensus [4ndash7] The reasons of inconsistent resultsare ascribed to the differences in the enrolled participantsobservation periods endpoints studied and particularly thepresence or absence of confounders Moreover the time-varying nature or trajectory of serum uric acid (UA) has beencompletely neglected in the previous study methods and therisk of serum UA may be too subtle to be independentlydetected in the existence of highly influential risk factorssuch as proteinuria and hypertension [8]We recently showedthat the effect of serum UA in the follow-up influenced the

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 3759153 14 pageshttpsdoiorg10115520173759153

2 Oxidative Medicine and Cellular Longevity

incipient ESRD by a propensity score analysis and that serumUA should be kept less than 65mgdL to inhibit the renaloutcome [8]

Several interventional randomized controlled trials(RCT) revealed the significant inhibition of decline inestimated glomerular filtration rate (eGFR) by allopurinola xanthine oxidase (XO) inhibitor but the small number ofparticipants and short observation duration hampered thedefinite conclusion [9ndash11] Only one recent study successfullyshowed that allopurinol inhibited reaching renal endpointsof doubling of serum creatinine and incidence of ESRDby the time-to-event analysis [12] A double-blind RCTrecruiting more than 400 participants is under way in Japanusing a recently developed novel XO inhibitor febuxostat[13]

Together with clinical evidence experimental studiesproviding mechanistic insights of UA-caused kidney injuriesare necessary A rat model receiving oxonic acid an inhibitorof uricase has been widely used to study the patho-physiological roles of hyperuricemia [14ndash17] These studiesprovided insights into the mechanisms for cardiovascularinjury associated with hyperuricemia and demonstrated thatUA directly causes vascular injury and hypertension viacrystal-independent mechanisms [15 16 18] Importantlyalthough UA is a strong antioxidant in the plasma [19]hyperuricemia accelerates target organ damage through theprooxidant property of UA [20] In vascular endothelial cellsoxidative stress associated with high UA levels decreasedendothelial nitric oxide leading to endothelial dysfunc-tion [21] Recent studies also demonstrated the role ofoxidative stress in systemic hypertension associated withhyperuricemia [17 21] Thus far however whether hyper-uricemia causes kidney damage solely via vascular injuryremains unclear Of note previous studies demonstratedthat hyperuricemia aggravates proteinuria in the rat remnantkidney model [22] although the mechanisms remain largelyobscure

Glomerular visceral epithelial cells or podocytes arepresent outside the glomerular basement membrane andserve as the filtration barrier to prevent the leak of plasmaproteins into the urine These cells constitute characteristicinterdigitating foot processes which are connected to eachother by the slit diaphragm proteins such as podocin andother molecules [23ndash25] Because the normal formationof podocyte slit diaphragms is the integral part of theglomerular permselectivity its dysregulation constitutes amajor cause of pathological proteinuria [26] Interestinglyaccumulating data revealed that podocytopathy plays a fun-damental role in kidney diseases associated with metabolicdisorders such as diabetic kidney disease salt-sensitivehypertension and obesity-related glomerulopathy [27ndash31]However despite the possible link between hyperuricemiaand CKD little is known on the role of hyperuricemia inmodulating podocyte function Thus we set out to exam-ine the increase in albuminuria and the involvement ofpodocytes in the kidney injury caused by experimental hype-ruricemia in conjunction with the involvement of oxidativestress

2 Materials and Methods

21 Animal Experiments All animal experiments were per-formed in accordance with the Institute Animal Care andUse Committee of the Teikyo University (Teikyo UniversitySchool of Medicine Animal Ethics Committee 14-035)Male Sprague-Dawley rats weighing 200 g were purchasedfrom Sankyo Lab (Tokyo Japan) Rats were divided intotwo groups after body weight urine collection and bloodpressure measurement One group received standard diet(CRF1 Oriental Yeast Tokyo Japan) (119899 = 13) whereasthe other group received oxonic acid (Sigma St Louis MOUSA)mixed in the diet (2 g100 g chow the dose was decidedaccording to previous studies) (119899 = 12) [18] Body weightand blood pressure were measured at 4 and 8 weeks Urinewas collected for 24 hours using individual metabolic cagesat 4 and 8 weeks At 8 weeks animals were euthanized underanesthesia using inhaled isoflurane

In another set of experiments rats received oxonic acid(2 g100 g chow) and tempol a superoxide dismutasemimetic(119899 = 8) Tempol was administered via drinking water at aconcentration of 1mmolL which is shown to be effectivein several rodent models [32 33] Rats that received oxonicacid and normal water (119899 = 7) were used as controls toevaluate the protective effects of tempol After blood pressuremeasurement and urine collection animals were euthanizedat 8 weeks

Systolic blood pressure of conscious rats wasmeasured bythe tail-cuffmethod Blood samples were obtained by cardiacpuncture Kidneys were removed snap-frozen and stored atminus80∘C until use Urinary albumin levels were measured byELISA (SRL Tokyo Japan) SDS-PAGE analysis of the urinewas performed as described previously [34]

Serum UA concentrations were determined using high-performance liquid chromatograph equipped with a UVspectrophotometric detector (Prominence Shimadzu KyotoJapan) UA standard was dissolved in water by adding2mmolL ammonium hydroxide solution (final concentra-tion of 5mgdL) Serum sampleswere centrifuged andfilteredthrough a Millipore filter (022 120583m pore size DarmstadtGermany) Samples were injected onto a Wakosil GP-N6column (15 times 46mm ID) with mobile phase of 98 (vv)02molL sodium phosphate buffer pH60 and 2 (vv) ace-tonitrile at a flow rate of 05mLmin Under these conditionstypical retention time for uric acid (detected at 284 nm) was368min

22 Immunohistochemistry and Quantification Kidney tis-sues were fixed in 4 paraformaldehyde in PBS at 4∘CTissues were incubated in 30 sucrose in PBS overnightat 4∘C and mounted in OCT (Tissue-Tek Tokyo Japan)for sectioning [34] After blocking tissue sections werestained with the indicated primary antibodies and affinity-purified secondary antibodies-conjugated HRP (DAKOGlostrup Denmark) Primary antibodies used included anti-bodies against 120572SMA (Sigma) desmin (DAKO GlostrupDenmark) podocin (Abcam Cambridge MA USA) and8-hydroxy-21015840-deoxyguanosine (8OHdG) (JaICA ShizuokaJapan) Quantitative analysis of afferent arterioles was

Oxidative Medicine and Cellular Longevity 3

Table 1 Biological parameters in control and hyperuricemic (HUA) rats at 8 weeks

Control HUABody weight g 4592 plusmn 255 4533 plusmn 385Blood pressure mmHg 1246 plusmn 50 1416 plusmn 27lowastlowast

Urine volume mL 133 plusmn 70 144 plusmn 63Urinary albumin 120583gday 1770 plusmn 1024 5437 plusmn 3171lowastlowast

Serum UA mgdL 067 plusmn 042 132 plusmn 035lowastlowast

Serum Cr mgdL 034 plusmn 002 033 plusmn 002Values are means plusmn SD lowastlowast119875 lt 001 versus control

performed as previously described [16] For afferent arteri-oles vessels with internal elastic lamina adjacent to glomeruliwere selected Arcuate arteries were identified by the locationat the border of renal cortex and medulla Areas positive for120572SMA in the cross section of these vessels were quantitatedusing NanoZoomer (Hamamatsu Photonics HamamatsuJapan) and Aperio ImageScope (Leica Buffalo Grove ILUSA) For quantification of desmin podocin and 8OHdG inthe glomeruli the percentage of positive area was determinedas positive pixels per total pixels in a glomerulus using ImageScope software For each rat 20 glomeruli were randomlyanalyzed

23 Transmission Electron Microscopy Ultramicrostructureof the glomeruli was observed by transmission electronmicroscopy Small pieces of cortex were fixed in 25glutaraldehyde dehydrated through graded ethanol andpropylene oxide and embedded in Epon 812 using standardprocedures Ultrathin sections were stained with uranylacetate and with Reynolds lead citrate The specimens wereobserved using Hitachi transmission electronmicroscope H-7650 (Hitachi Science Systems Ltd Hitachinaka Japan)

24 XO Activity Measurement in the Kidney Cortex XOactivity was measured as xanthine oxidoreductase (XOR)activity using amethoddescribed previously [35] In brief thekidney cortex was homogenized in phosphate buffered saline(pH 74) containing protease inhibitor cocktail (Roche BaselSwitzerland) and centrifuged at 20000timesg 4∘C for 20minThe kidney homogenates were added to mixture containing[15N2] xanthine (04mmolL) NAD+ (04mmolL) and

oxonate (0013mmolL) in 20mmolL Tris buffer (pH 85)and were incubated at 37∘C for 30min Subsequently themixtures were mixed with 500 120583L of methanol containing[13C2 15N2] uric acid as internal standard and centrifuged

at 20000timesg for 10min at 4∘C The supernatants were trans-ferred to new tubes and dried using centrifugal evaporatorThe residues were reconstituted with 150120583L of distilled waterfiltered through an ultrafiltration membrane (Amicon Ultra05 centrifugal filter devices 3K Millipore Merck KGaADarmstadt Germany) and the [15N

2] uric acid production

was measured with LCMS (LTQ-Orbitrap Thermo FisherScientific Waltham MA USA) Each activity was expressedas [15N

2] uric acid production nmoLminmg protein

25 Statistical Analysis All data are continuous variables andthus expressed as mean plusmn standard deviation (SD) Based onthe distribution of the data the parametric statistics wereutilized Unpaired 119905-test was used for comparisons betweentwo groups Correlation of between parameters was analyzedby Pearsonrsquos correlation test A value of 119875 lt 005 was consid-ered statistically significant

3 Results and Discussion

31 Blood Pressure Elevation and Renal Vasculopathy inExperimental Hyperuricemia To evaluate the mechanismwhereby hyperuricemia impairs kidney function we orallyadministered oxonic acid (OA) the uricase inhibitor to maleSprague-Dawley rats [18] This model causes hyperuricemiawithout increasing purine metabolism and therefore intheory without increasing XO activity As shown in Table 1and Figure 1(a) OA successfully increased serumUA levels at8 weeks compared with the control group (119875 = 0002) Bodyweight was similar between the two groups (Figure 1(b))indicating that OA did not affect food and water intakeConsistent with the previous report [18] hyperuricemiacaused a moderate increase in systolic blood pressure whencompared to the control group at 8 weeks (119875 lt 0001) (Table 1and Figure 1(c))

Clinical and experimental studies indicated that hype-ruricemia is associated with renal arteriolopathy [15 1836] We next examined whether hyperuricemia inducesarteriopathy as well as arteriolopathy Kidney sections fromcontrol and hyperuricemic rats were stained with 120572-smoothmuscle actin (120572SMA) a marker for vascular smooth mus-cle cells (VSMC) to evaluate vascular hypertrophy at thelevels of afferent arterioles and arcuate arteries As shownin Figure 2(a) hyperuricemia caused thickening of afferentarterioles in hyperuricemic rats compared to control rats(119875 = 0043) The vascular changes were not limited to thearterioles but were also present in the medium-sized arteriesas demonstrated by the increased 120572SMA staining in renalarcuate arteries (119875 = 0004 Figure 2(b)) In addition to theconfirmation of the finding of the arterioles [16 36] we coulddemonstrate the involvement of medium-sized arteries in thevasculopathy induced by hyperuricemia

32 Podocyte Injury Is Involved in Hyperuricemic Rats Giventhe experimental evidence that hyperuricemia facilitates the


20

15

10

05

00HUACtrl

Seru

m U

A (m

gdL

)lowastlowast

(a)

600

400

0

200

HUACtrl

0 4 8

Body

wei

ght (

g)

(week)

(b)160

140

120

100

80

Systo

lic b

lood

pre

ssur

e (m

mH

g)

lowastlowast

HUACtrl

0 4 8

(week)

(c)

Figure 1 Blood pressure elevation in oxonic acid-treated hyperuricemic rats (a) Serum uric acid (UA) levels were measured by high-performance liquid chromatography at 8 weeks in control rats (Ctrl) and hyperuricemic rats (HUA) receiving 2 oxonic acid (b) Bodyweight in control (Ctrl) and hyperuricemic (HUA) rats Body weight did not differ throughout the experiment (c) Systolic blood pressurewas significantly higher in HUA group than Ctrl at 8 weeks Data are expressed as mean plusmn SD 119899 = 12 or 13 per group lowastlowast119875 lt 001

progression of kidney injury in the rat remnant kidneymodel [22] we next determined whether hyperuricemiaper se (ie without nephrectomy) causes kidney damageAlthough the serum creatinine levels did not significantlydiffer between control and hyperuricemic rats (Table 1 119875 =0804) albuminuria progressively increased in hyperuricemic

rats (Figure 3(a) and Table 1) the urinary albumin levels weresignificantly higher at as early as 4 weeks (119875 = 0049) and fur-ther increased at 8 weeks (119875 = 00015) SDS-PAGE analysisof urine obtained from hyperuricemic rats resembled theelectrophoresis pattern of serum proteins consistent withnonselective glomerular proteinuria (Figure 3(b))


lowast

120572-S

MA

pos

itive

area

(120583m2)

HUACtrl

400

0

300

200

100

HUACtrl

(a)lowastlowast

120572-S

MA

pos

itive

area

(120583m2)

5000

10000

15000

20000

HUACtrl

HUACtrl

(b)

Figure 2Thickening of afferent arterioles and arcuate arteries in hyperuricemic rats (a and b) Rat kidney sections were stained for 120572-smoothmuscle actin (120572-SMA) to evaluate the thickening of afferent arterioles (a) and arcuate arteries (b) Bars represent 50 120583m Bar graphs show theresults of quantitation Data are expressed as mean plusmn SD 119899 = 6 for each group lowast119875 lt 005 lowastlowast119875 lt 001

By forming foot processes and slit diaphragms podocytesplay central roles to prevent albuminuria in a normal stateTo determine the cause of increased urinary albumin inthis model we analyzed the involvement of podocytesInterestingly desmin a sensitive podocyte injury [27 37]was upregulated in podocytes of hyperuricemic rats butnot in those of control rats (Figures 3(c) and 3(d)) More-over immunostaining of the slit diaphragm componentpodocin revealed that it was significantly decreased in hype-ruricemic rats (Figure 3(e)) Consistent with these findingsa structural analysis using transmission electron microscopy

demonstrated the occasional retraction of the podocyte footprocesses in hyperuricemic rats (Figure 4) Podocytes fromhyperuricemic rats also showed a sign of microvillus trans-formation (Figure 4(c)) indicating podocyte damage [38]These data are consistent with the immunohistochemicalanalysis and demonstrate that podocytopathy underlies theincrease in albuminuria in the hyperuricemic model

33 Role of Oxidative Stress in Vasculopathy and PodocyteInjury Induced by Hyperuricemia The above data indicatethat podocyte is involved in the kidney injury associated


1000

600

800

400

0

200

0 4 8

(week)

Urin

ary

albu

min

(120583g

day)

HUACtrl

lowastlowast

lowast

(a)

(kDa)

10

15

20

25

37

50

75

100

150

250

MWUrineSerum

(b)

HUACtrl

(c)

HUA

HUA

Ctrl0

10

5

15

20

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ive a

rea

glom

erul

us (

)

lowastlowast

Ctrl

(d)

Figure 3 Continued


HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)

Figure 3 Involvement of podocyte injury in hyperuricemic rats (a) Urinary albumin excretion measured at 8 weeks in control (Ctrl) andhyperuricemic (HUA) rats (b) SDS-PAGE analysis of serum and urine from hyperuricemic rats (c) and (d) Representative micrographsof immunostaining for desmin a marker for podocyte injury at low (c) and high (d) magnifications Bar graphs show the quantitativeevaluation of desmin staining in the glomeruli (e) Representativemicrographs of immunostaining for podocin a component of the podocyteslit diaphragm Bar graphs show the quantitative evaluation of podocin staining in the glomeruli Data are expressed as mean plusmn SD 119899 = 12or 13 for (a) 119899 = 6 or 7 for (d) and 119899 = 3 for (e) Bars represent 50120583m (c) and 10120583m (d e) lowast119875 lt 005 lowastlowast119875 lt 001 Arrows indicate increaseddesmin staining in podocytes

with hyperuricemia resulting in increased urinary albuminexcretion Multiple lines of evidence demonstrate the impor-tance of oxidative stress in podocyte dysfunction especiallywhen associated with endocrine and metabolic disorders[27 31 39] To elucidate the possible mechanisms under-lying podocyte injury in hyperuricemic rats we examinedthe expression of oxidative stress markers in our modelRemarkably urinary 8OHdG levels were as much as 4-foldhigher in hyperuricemic rats compared with control rats(1130 plusmn 466 ngday in hyperuricemic group versus 281 plusmn80 ngday in control group 119875 lt 0001) (Figure 5(a)) More-over the immunostaining of the kidney cortex revealed that8OHdG staining was upregulated in glomerular cells includ-ing podocytes (Figures 5(b) and 5(c))

Correlation analysis revealed that albuminuria correlatedwith urinary 8OHdG levels (1198772 = 049 Figure 6(a)) Albu-minuria was also positively correlated with blood pressurelevels although to a lesser extent (1198772 = 029 Figure 6(b)) Wefurther evaluated the relationship between urinary 8OHdGand the indices of podocyte injury and vascular remodelingOf note the index of podocytopathy was highly correlatedwith urinary 8OHdG levels (1198772 = 080 Figure 6(c)) furthersupporting the involvement of reactive oxygen species inpodocyte damage Urinary 8OHdG levels also correlatedwiththe degree of arteriolopathy but to a lesser extent (1198772 = 043Figure 6(d))

Previous studies reported that tempol a superoxide dis-mutase mimetic successfully ameliorated vascular damageand blood pressure elevation in experimental hyperuricemia

[17 40] To test whether podocyte injury in our model wasinduced by similar mechanisms we administered tempol tohyperuricemic rats Consistent with previous reports [17 40]systolic blood pressure levels were significantly lower in thehyperuricemia plus tempol group than in the hyperuricemiagroup (Figure 7(a)) Quantitative analysis using 120572SMA stain-ing demonstrated that tempol also ameliorated the thick-ening of the afferent arterioles (Figure 7(b)) Interestinglyhowever urinary albumin levels were not altered by tempol(Figure 7(c)) despite the reduced blood pressure and theprevention of vasculopathy This discrepancy indicates thathyperuricemia-induced podocyte injury and the resultantalbuminuria may occur independently of vascular dysfunc-tion and hypertension

To investigate the role of XO activity in the presentmodel we compared XOR activity in the kidney of controland hyperuricemic rats As shown in Figure 8 XOR activitywas not elevated but rather tended to be decreased inhyperuricemic rats compared with control rats

34 Discussion In this study we demonstrated that theexperimental hyperuricemia induced by uricase inhibitionis associated with podocyte injury and significant albu-minuria Podocyte injuries were confirmed by the increasein desmin expression in podocytes and by slit membraneabnormalities including decreased podocin expression andaugmented foot process effacement assessed by transmissionelectron microscopy Podocyte injury can be related toactivation of cellular oxidative stress given that the degrees


Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)

Figure 4 Podocyte injury in hyperuricemic rats is confirmed by electron microscopy Transmission electron micrographs of podocyte footprocess in the glomeruli of indicated animals Podocytes in the kidney from hyperuricemic rats (HUA) showed foot process effacement(arrowheads) and microvillus transformation (MV) These changes were less evident in the control (Ctrl) group Bars represent 5 120583m

of albuminuria and desmin staining correlated with urinary8OHdG excretion Of interest tempol ameliorated highblood pressure and vascular remodeling corroborating withthe previous reports [17 40] but failed to reduce albu-minuria Although the previous studies did not investigatethe relation between albuminuria and tempol the presentresults may indicate that arteriolopathy and podocytopa-thy occur via distinct mechanisms in hyperuricemic stateA possible pathological mechanism may be attributed tomitochondrial alterations and decreased intracellular ATPconcentrations [21] but more studies are needed to explorethe hyperuricemia-induced podocyte injury independentlyof superoxide-mediated mechanism

The involvement of oxidative stress in hyperuricemia-induced kidney injurywas recently emphasized in the contextof XO inhibition with an advent of novel XO inhibitors suchas febuxostat and topiroxostat [20]The activity of XO gener-ates superoxide in the cells leading to cell damage which canbe abrogated by treatment with XO inhibitors [35 41] In fact

topiroxostat significantly reduced urinary albumin excretionby 33 of the baseline at 22 weeks in patients with stage3 CKD [13] Similarly febuxostat treatment of patients withstage 3 CKD could significantly decrease the albuminuriaand proteinuria in 12 weeks [42] In theory however itwas supposed that our hyperuricemic rat model induced byuricase inhibitor may not necessarily be associated with theincrease in XO activity Indeed XOR activity measured inthe kidney was not elevated or rather tended to decreaseprobably due to the product inhibition in hyperuricemicmodel rats [43] Therefore UA itself but not XO activitymay play a critical role in the kidney injuries seen in thepresent study Nonetheless given that kidneys are composedof many different cell types our data do not exclude thepossibility that XO activity is increased in specific cell typesincluding podocytes It is generally accepted that extracellular(circulating) UA is the most abundant aqueous antioxidantin humans and serves as the major free radical scavenger inplasma [19] The extracellular UA is particularly effective in


HUACtrl0

500

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1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

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(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)

Figure 5 Involvement of oxidative stress in the glomerular injury in hyperuricemic rats (a) Urinary levels of 8-hydroxy-21015840-deoxyguanosine(8OHdG) were measured in control (Ctrl) and hyperuricemic rats (HUA) (b) Staining of 8OHdG in the glomeruli Arrows indicate theenhancement of the staining Bar represents 50120583m (c) Quantitative evaluation of 8OHdG staining in the glomeruli 8OHdG-positive nucleiwere counted as percentage of total glomerular nuclei Data are expressed as mean plusmn SD 119899 = 12 for (a) 119899 = 7 for (c) lowast119875 lt 005 lowastlowast119875 lt 001

quenching hydroxyl superoxide and peroxynitrite radicalsand may serve a protective physiological role by preventinglipid peroxidation [19] On the other hand UA loses itsradical scavenging activity under hydrophobic conditions[44] and can become a strong prooxidant within the cellsIndeed recent studies indicate that UA once enters intointracellular compartment increases reactive oxygen species[21 45 46] Consistent with these data we found that hype-ruricemia caused glomerular injury which was associatedwith increased 8OHdG levels Taken all together UA mayserve either a scavenger or a prooxidant depending on the

biological milieu In addition two distinct pathophysiolog-ical scenarios by UA itself and by XO-mediated chain ofevents may underlie the pathophysiology of cellular damageassociated with hyperuricemia

Another question arises how intracellular UA is increasedin response to the increase in serum UA One possibilityis that UA is taken up by a sort of urate transporter(s)Since the discovery of the first urate transporter URAT1 inthe renal proximal tubular cells [47] accumulating evidenceindicates that URAT1 is also present in other cell types suchas vascular smoothmuscle cells (VSMC) [48] and endothelial


00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)

Figure 6 Correlation of different parameters in control and hyperuricemic rats (a and b) Urinary albumin levels correlated with urinary8OHdG levels (a) and systolic blood pressure (b) The coefficient of determination was 049 for (a) and 029 for (b) (c and d) Correlationbetween urinary 8OHdG levels and the degree of podocytopathy as determined by desmin-positive area (c) or the degree of arteriolopathyas determined by the afferent arteriole thickness (d)


HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

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(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)

Figure 7 Superoxide dismutase (SOD)mimic tempol ameliorated hypertension but did not reduce albuminuria in hyperuricemic rats (andashc)Systolic blood pressure (a) thickening of afferent arterioles as assessed by 120572-smooth muscle actin staining (b) and urinary albumin (c) inHUA rats with and without coadministration of tempol Data are expressed as mean plusmn SD 119899 = 7 for HUA and 8 for HUA + TEMP (a and c)119899 = 4 for (b) lowast119875 lt 005 NS not significant

cells [49] In VSMC UA enters the cells via URAT1 resultingin the activation of transcription factors and cytokinesincluding nuclear factor-120581B activator protein-1 and mono-cyte chemoattractant protein-1 ultimately leading to VSMCproliferation and vascular dysfunction [22 48] More recentdata indicate that UA can induce signaling in renal mesangialcells [50] and collecting duct cells [14] Thus it is possiblethat UA enters into glomerular podocytes leading to tissue

damage and resultant albuminuria in the setting of hyper-uricemia Anyhow future studies arewarranted to explore themechanisms whereby intracellular UA modulates podocytefunction

A potential limitation of our study is that we did notprovide detailed mechanisms of glomerular podocyte injuryobserved in our model Future studies using cell culture arenecessary to evaluate the causal role of UA and downstream


4000

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2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)

Figure 8 Xanthine oxidoreductase activity in the kidney Levels ofxanthine oxidoreductase activity weremeasured in the kidneys fromcontrol (Ctrl) and hyperuricemic rats (HUA)

signaling in podocyte damage Biological actions of oxonicacid other than uricase inhibition may also be consideredalthough it is widely used to study the effects of hyper-uricemia in rats

4 Conclusion

The present study demonstrates that hyperuricemia in ratsinduces albuminuria associated with podocyte injury Ourdata indicate that hyperuricemia can play a causal role inthe progression of CKD not only by promoting circulatorysystem abnormalities but also by increasing albuminuriaone of the most influential risk factors Therefore these dataindicate the importance of appropriately controlling serumUA to prevent decline in kidney function in patients withCKD

Disclosure

The funders had no role in study design execution ofthe experiment decision to publish or preparation of themanuscript

Competing Interests

The authors have declared that no competing interests exist

Acknowledgments

The authors thank Ms Hiromi Yamaguchi Ms EmikoOkuma-Kuribayashi and Ms Miyuki Fukazawa for theirexcellent technical support This study was supported in partby a Grant-in-Aid for Progressive Renal Diseases ResearchResearch on Rare and Intractable Disease from the Ministryof Health Labour and Welfare of Japan (to Shunya Uchida)Gout Research Foundation (to Shunya Uchida) and JSPSKAKENHI Grants 15H04837 (to Shigeru Shibata)

References

[1] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[2] A S Levey R Atkins J Coresh et al ldquoChronic kidney diseaseas a global public health problem approaches and initiativesmdasha position statement from Kidney Disease Improving GlobalOutcomesrdquo Kidney International vol 72 no 3 pp 247ndash2592007

[3] D S Keith G A Nichols C M Gullion J B Brown andD H Smith ldquoLongitudinal follow-up and outcomes among apopulation with chronic kidney disease in a large managed careorganizationrdquo Archives of Internal Medicine vol 164 no 6 pp659ndash663 2004

[4] W Yang D Xie A H Anderson et al ldquoAssociation of kidneydisease outcomes with risk factors for CKD findings from theChronic Renal Insufficiency Cohort (CRIC) studyrdquo AmericanJournal of Kidney Diseases vol 63 no 2 pp 236ndash243 2014

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

[6] L Li C Yang Y Zhao X Zeng F Liu and P Fu ldquoIs hype-ruricemia an independent risk factor for new-onset chronickidney disease a systematic review andmeta-analysis based onobservational cohort studiesrdquo BMC Nephrology vol 15 article122 2014

[7] T Kumagai T Ota Y Tamura W X Chang S Shibata and SUchida ldquoTime to target uric acid to retard CKD progressionrdquoClinical and Experimental Nephrology pp 1ndash11 2016

[8] S Uchida W X Chang T Ota et al ldquoTargeting uric acidand the inhibition of progression to end-stage renal diseasemdasha propensity score analysisrdquo PLoS ONE vol 10 no 12 ArticleID e0145506 2015

[9] T Kanji M Gandhi C M Clase and R Yang ldquoUrate loweringtherapy to improve renal outcomes in patients with chronickidney disease systematic review and meta-analysisrdquo BMCNephrology vol 16 article 58 2015

[10] R J Johnson T Nakagawa D Jalal L G Sanchez-Lozada D-H Kang and E Ritz ldquoUric acid and chronic kidney diseasewhich is chasing whichrdquo Nephrology Dialysis Transplantationvol 28 no 9 pp 2221ndash2228 2013

[11] B Bose S V Badve S S Hiremath et al ldquoEffects of uric acid-lowering therapy on renal outcomes a systematic review andmeta-analysisrdquoNephrology Dialysis Transplantation vol 29 no2 pp 406ndash413 2014

[12] M Goicoechea S Garcia DeVinuesa U Verdalles et al ldquoAllop-urinol and progression of CKD and cardiovascular eventsLong-term follow-up of a randomized clinical trialrdquo AmericanJournal of Kidney Diseases vol 65 no 4 pp 543ndash549 2015

[13] T Hosoya K Kimura S Itoh et al ldquoThe effect of febuxostatto prevent a further reduction in renal function of patients withhyperuricemia who have never had gout and are complicated bychronic kidney disease stage 3 study protocol for a multicenterrandomized controlled studyrdquoTrials vol 15 no 1 article no 262014

[14] W Xu Y Huang L Li et al ldquoHyperuricemia induces hyper-tension through activation of renal epithelial sodium channel(ENaC)rdquo Metabolism Clinical and Experimental vol 65 no 3pp 73ndash83 2016


[15] L G Sanchez-Lozada E Tapia J Santamarıa et al ldquoMild hype-ruricemia induces vasoconstriction and maintains glomerularhypertension in normal and remnant kidney ratsrdquo KidneyInternational vol 67 no 1 pp 237ndash247 2005

[16] M Mazzali J Kanellis L Han et al ldquoHyperuricemia inducesa primary renal arteriolopathy in rats by a blood pressure-independent mechanismrdquo American Journal of PhysiologymdashRenal Physiology vol 282 no 6 pp F991ndashF997 2002

[17] M Cristobal-Garcıa F E Garcıa-Arroyo E Tapia et al ldquoRenaloxidative stress induced by long-term hyperuricemia altersmitochondrial function and maintains systemic hypertensionrdquoOxidative Medicine and Cellular Longevity vol 2015 Article ID535686 8 pages 2015

[18] M Mazzali J Hughes Y-G Kim et al ldquoElevated uric acidincreases blood pressure in the rat by a novel crystal-inde-pendent mechanismrdquoHypertension vol 38 no 5 pp 1101ndash11062001

[19] W S Waring ldquoUric acid an important antioxidant in acuteischaemic strokerdquo QJM vol 95 no 10 pp 691ndash693 2002

[20] C J Chen J-M Lu and Q Yao ldquoHyperuricemia-relateddiseases and xanthine oxidoreductase (XOR) inhibitors anoverviewrdquoMedical Science Monitor vol 22 pp 2501ndash2512 2016

[21] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2012

[22] D-H Kang T Nakagawa L Feng et al ldquoA role for uric acid inthe progression of renal diseaserdquo Journal of the American Societyof Nephrology vol 13 no 12 pp 2888ndash2897 2002

[23] V Ruotsalainen P Ljungberg J Wartiovaara et al ldquoNephrinis specifically located at the slit diaphragm of glomerularpodocytesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 96 no 14 pp 7962ndash7967 1999

[24] M Kestila U Lenkkeri M Mannikko et al ldquoPositionallycloned gene for a novel glomerular proteinmdashnephrinmdashismutated in congenital nephrotic syndromerdquoMolecular Cell vol1 no 4 pp 575ndash582 1998

[25] N Boute O Gribouval S Roselli et al ldquoNPHS2 encoding theglomerular protein podocin is mutated in autosomal recessivesteroid-resistant nephrotic syndromerdquo Nature Genetics vol 24no 4 pp 349ndash354 2000

[26] M Nagata ldquoPodocyte injury and its consequencesrdquo KidneyInternational vol 89 no 6 pp 1221ndash1230 2016

[27] S Shibata M Nagase S Yoshida H Kawachi and T FujitaldquoPodocyte as the target for aldosterone roles of oxidative stressand Sgk1rdquo Hypertension vol 49 no 2 pp 355ndash364 2007

[28] S Shibata S Mu H Kawarazaki et al ldquoRac1 GTPase inrodent kidneys is essential for salt-sensitive hypertension via amineralocorticoid receptor-dependent pathwayrdquoThe Journal ofClinical Investigation vol 121 no 8 pp 3233ndash3243 2011

[29] K Reidy H M Kang T Hostetter and K Susztak ldquoMolecularmechanisms of diabetic kidney diseaserdquoThe Journal of ClinicalInvestigation vol 124 no 6 pp 2333ndash2340 2014

[30] M Nagase S Yoshida S Shibata et al ldquoEnhanced aldosteronesignaling in the early nephropathy of rats with metabolicsyndrome possible contribution of fat-derived factorsrdquo Journalof the American Society of Nephrology vol 17 no 12 pp 3438ndash3446 2006

[31] V D DrsquoAgati A Chagnac A P J de Vries et al ldquoObesity-related glomerulopathy clinical and pathologic characteristics

and pathogenesisrdquoNature Reviews Nephrology vol 12 no 8 pp453ndash471 2016

[32] C G Schnackenberg and C S Wilcox ldquoTwo-week administra-tion of tempol attenuates both hypertension and renal excretionof 8-Iso prostaglandin F(2120572)rdquo Hypertension vol 33 no 1 part2 pp 424ndash428 1999

[33] B ElmedalMY deDamM JMulvany andU Simonsen ldquoThesuperoxide dismutase mimetic tempol blunts right ventricularhypertrophy in chronic hypoxic ratsrdquoBritish Journal of Pharma-cology vol 141 no 1 pp 105ndash113 2004

[34] S Shibata M Nagase S Yoshida et al ldquoModification of miner-alocorticoid receptor function by Rac1 GTPase implication inproteinuric kidney diseaserdquo Nature Medicine vol 14 no 12 pp1370ndash1376 2008

[35] 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

[36] KKohaguraMKochi TMiyagi et al ldquoAn association betweenuric acid levels and renal arteriolopathy in chronic kidneydisease a biopsy-based studyrdquo Hypertension Research vol 36no 1 pp 43ndash49 2013

[37] E Yaoita K Kawasaki T Yamamoto and I Kihara ldquoVariableexpression of desmin in rat glomerular epithelial cellsrdquo Ameri-can Journal of Pathology vol 136 no 4 pp 899ndash908 1990

[38] M Hara T Yanagihara I Kihara K Higashi K Fujimotoand T Kajita ldquoApical cell membranes are shed into urine frominjured podocytes a novel phenomenon of podocyte injuryrdquoJournal of the American Society of Nephrology vol 16 no 2 pp408ndash416 2004

[39] A Whaley-Connell V G Demarco G Lastra et al ldquoInsulinresistance oxidative stress and podocyte injury role of rosu-vastatin modulation of filtration barrier injuryrdquo AmericanJournal of Nephrology vol 28 no 1 pp 67ndash75 2008

[40] L G Sanchez-Lozada V Soto E Tapia et al ldquoRole of oxidativestress in the renal abnormalities induced by experimental hype-ruricemiardquo American Journal of PhysiologymdashRenal Physiologyvol 295 no 4 pp F1134ndashF1141 2008

[41] H Tsuda N Kawada J-Y Kaimori et al ldquoFebuxostat sup-pressed renal ischemia-reperfusion injury via reduced oxidativestressrdquo Biochemical and Biophysical Research Communicationsvol 427 no 2 pp 266ndash272 2012

[42] K Tanaka M Nakayama M Kanno et al ldquoRenoprotectiveeffects of febuxostat in hyperuricemic patients with chronickidney disease a parallel-group randomized controlled trialrdquoClinical and Experimental Nephrology vol 19 no 6 pp 1044ndash1053 2015

[43] R Radi S Tan E Prodanov R A Evans and D A ParksldquoInhibition of xanthine oxidase by uric acid and its influence onsuperoxide radical productionrdquo Biochimica et Biophysica Acta(BBA) vol 1122 no 2 pp 178ndash182 1992

[44] S Muraoka and T Miura ldquoInhibition by uric acid of freeradicals that damage biological moleculesrdquo Pharmacology andToxicology vol 93 no 6 pp 284ndash289 2003

[45] D Kadowaki S Sakaguchi Y Miyamoto et al ldquoDirect radi-cal scavenging activity of benzbromarone provides beneficialantioxidant properties for hyperuricemia treatmentrdquo Biologicaland Pharmaceutical Bulletin vol 38 no 3 pp 487ndash492 2015

[46] H Xie J Sun Y Chen M Zong S Li and Y Wang ldquoEGCGattenuates uric acid-induced inflammatory and oxidativestress responses by medicating the notch pathwayrdquo Oxidative


Medicine and Cellular Longevity vol 2015 Article ID 21483610 pages 2015

[47] A Enomoto H Kimura A Chairoungdua et al ldquoMolecularidentification of a renal urate-anion exchanger that regulatesblood urate levelsrdquoNature vol 417 no 6887 pp 447ndash452 2002

[48] K L Price Y Y Sautin D A Long et al ldquoHuman vascularsmooth muscle cells express a urate transporterrdquo Journal of theAmerican Society of Nephrology vol 17 no 7 pp 1791ndash17952006

[49] S Sugihara I Hisatome M Kuwabara et al ldquoDepletion ofuric acid due to SLC22A12 (URAT1) loss-of-function mutationcauses endothelial dysfunction in hypouricemiardquo CirculationJournal vol 79 no 5 pp 1125ndash1132 2015

[50] Y Zhuang Q Feng G Ding et al ldquoActivation of ERK12 byNADPH oxidase-originated reactive oxygen species mediatesuric acid-induced mesangial cell proliferationrdquo American Jour-nal of PhysiologymdashRenal Physiology vol 307 no 4 pp F396ndashF406 2014

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


incipient ESRD by a propensity score analysis and that serumUA should be kept less than 65mgdL to inhibit the renaloutcome [8]

Several interventional randomized controlled trials(RCT) revealed the significant inhibition of decline inestimated glomerular filtration rate (eGFR) by allopurinola xanthine oxidase (XO) inhibitor but the small number ofparticipants and short observation duration hampered thedefinite conclusion [9ndash11] Only one recent study successfullyshowed that allopurinol inhibited reaching renal endpointsof doubling of serum creatinine and incidence of ESRDby the time-to-event analysis [12] A double-blind RCTrecruiting more than 400 participants is under way in Japanusing a recently developed novel XO inhibitor febuxostat[13]

Together with clinical evidence experimental studiesproviding mechanistic insights of UA-caused kidney injuriesare necessary A rat model receiving oxonic acid an inhibitorof uricase has been widely used to study the patho-physiological roles of hyperuricemia [14ndash17] These studiesprovided insights into the mechanisms for cardiovascularinjury associated with hyperuricemia and demonstrated thatUA directly causes vascular injury and hypertension viacrystal-independent mechanisms [15 16 18] Importantlyalthough UA is a strong antioxidant in the plasma [19]hyperuricemia accelerates target organ damage through theprooxidant property of UA [20] In vascular endothelial cellsoxidative stress associated with high UA levels decreasedendothelial nitric oxide leading to endothelial dysfunc-tion [21] Recent studies also demonstrated the role ofoxidative stress in systemic hypertension associated withhyperuricemia [17 21] Thus far however whether hyper-uricemia causes kidney damage solely via vascular injuryremains unclear Of note previous studies demonstratedthat hyperuricemia aggravates proteinuria in the rat remnantkidney model [22] although the mechanisms remain largelyobscure

Glomerular visceral epithelial cells or podocytes arepresent outside the glomerular basement membrane andserve as the filtration barrier to prevent the leak of plasmaproteins into the urine These cells constitute characteristicinterdigitating foot processes which are connected to eachother by the slit diaphragm proteins such as podocin andother molecules [23ndash25] Because the normal formationof podocyte slit diaphragms is the integral part of theglomerular permselectivity its dysregulation constitutes amajor cause of pathological proteinuria [26] Interestinglyaccumulating data revealed that podocytopathy plays a fun-damental role in kidney diseases associated with metabolicdisorders such as diabetic kidney disease salt-sensitivehypertension and obesity-related glomerulopathy [27ndash31]However despite the possible link between hyperuricemiaand CKD little is known on the role of hyperuricemia inmodulating podocyte function Thus we set out to exam-ine the increase in albuminuria and the involvement ofpodocytes in the kidney injury caused by experimental hype-ruricemia in conjunction with the involvement of oxidativestress

2 Materials and Methods

21 Animal Experiments All animal experiments were per-formed in accordance with the Institute Animal Care andUse Committee of the Teikyo University (Teikyo UniversitySchool of Medicine Animal Ethics Committee 14-035)Male Sprague-Dawley rats weighing 200 g were purchasedfrom Sankyo Lab (Tokyo Japan) Rats were divided intotwo groups after body weight urine collection and bloodpressure measurement One group received standard diet(CRF1 Oriental Yeast Tokyo Japan) (119899 = 13) whereasthe other group received oxonic acid (Sigma St Louis MOUSA)mixed in the diet (2 g100 g chow the dose was decidedaccording to previous studies) (119899 = 12) [18] Body weightand blood pressure were measured at 4 and 8 weeks Urinewas collected for 24 hours using individual metabolic cagesat 4 and 8 weeks At 8 weeks animals were euthanized underanesthesia using inhaled isoflurane

In another set of experiments rats received oxonic acid(2 g100 g chow) and tempol a superoxide dismutasemimetic(119899 = 8) Tempol was administered via drinking water at aconcentration of 1mmolL which is shown to be effectivein several rodent models [32 33] Rats that received oxonicacid and normal water (119899 = 7) were used as controls toevaluate the protective effects of tempol After blood pressuremeasurement and urine collection animals were euthanizedat 8 weeks

Systolic blood pressure of conscious rats wasmeasured bythe tail-cuffmethod Blood samples were obtained by cardiacpuncture Kidneys were removed snap-frozen and stored atminus80∘C until use Urinary albumin levels were measured byELISA (SRL Tokyo Japan) SDS-PAGE analysis of the urinewas performed as described previously [34]

Serum UA concentrations were determined using high-performance liquid chromatograph equipped with a UVspectrophotometric detector (Prominence Shimadzu KyotoJapan) UA standard was dissolved in water by adding2mmolL ammonium hydroxide solution (final concentra-tion of 5mgdL) Serum sampleswere centrifuged andfilteredthrough a Millipore filter (022 120583m pore size DarmstadtGermany) Samples were injected onto a Wakosil GP-N6column (15 times 46mm ID) with mobile phase of 98 (vv)02molL sodium phosphate buffer pH60 and 2 (vv) ace-tonitrile at a flow rate of 05mLmin Under these conditionstypical retention time for uric acid (detected at 284 nm) was368min

22 Immunohistochemistry and Quantification Kidney tis-sues were fixed in 4 paraformaldehyde in PBS at 4∘CTissues were incubated in 30 sucrose in PBS overnightat 4∘C and mounted in OCT (Tissue-Tek Tokyo Japan)for sectioning [34] After blocking tissue sections werestained with the indicated primary antibodies and affinity-purified secondary antibodies-conjugated HRP (DAKOGlostrup Denmark) Primary antibodies used included anti-bodies against 120572SMA (Sigma) desmin (DAKO GlostrupDenmark) podocin (Abcam Cambridge MA USA) and8-hydroxy-21015840-deoxyguanosine (8OHdG) (JaICA ShizuokaJapan) Quantitative analysis of afferent arterioles was





















20

15

10

05

00HUACtrl

Seru

m U

A (m

gdL

)lowastlowast

(a)

600

400

0

200

HUACtrl

0 4 8

Body

wei

ght (

g)

(week)

(b)160

140

120

100

80

Systo

lic b

lood

pre

ssur

e (m

mH

g)

lowastlowast

HUACtrl

0 4 8

(week)

(c)





lowast

120572-S

MA

pos

itive

area

(120583m2)

HUACtrl

400

0

300

200

100

HUACtrl

(a)lowastlowast

120572-S

MA

pos

itive

area

(120583m2)

5000

10000

15000

20000

HUACtrl

HUACtrl

(b)






1000

600

800

400

0

200

0 4 8

(week)

Urin

ary

albu

min

(120583g

day)

HUACtrl

lowastlowast

lowast

(a)

(kDa)

10

15

20

25

37

50

75

100

150

250

MWUrineSerum

(b)

HUACtrl

(c)

HUA

HUA

Ctrl0

10

5

15

20

Posit

ive a

rea

glom

erul

us (

)

lowastlowast

Ctrl

(d)

Figure 3 Continued


HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)









Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































20

15

10

05

00HUACtrl

Seru

m U

A (m

gdL

)lowastlowast

(a)

600

400

0

200

HUACtrl

0 4 8

Body

wei

ght (

g)

(week)

(b)160

140

120

100

80

Systo

lic b

lood

pre

ssur

e (m

mH

g)

lowastlowast

HUACtrl

0 4 8

(week)

(c)





lowast

120572-S

MA

pos

itive

area

(120583m2)

HUACtrl

400

0

300

200

100

HUACtrl

(a)lowastlowast

120572-S

MA

pos

itive

area

(120583m2)

5000

10000

15000

20000

HUACtrl

HUACtrl

(b)






1000

600

800

400

0

200

0 4 8

(week)

Urin

ary

albu

min

(120583g

day)

HUACtrl

lowastlowast

lowast

(a)

(kDa)

10

15

20

25

37

50

75

100

150

250

MWUrineSerum

(b)

HUACtrl

(c)

HUA

HUA

Ctrl0

10

5

15

20

Posit

ive a

rea

glom

erul

us (

)

lowastlowast

Ctrl

(d)

Figure 3 Continued


HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)









Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

120572-S

MA

pos

itive

area

(120583m2)

HUACtrl

400

0

300

200

100

HUACtrl

(a)lowastlowast

120572-S

MA

pos

itive

area

(120583m2)

5000

10000

15000

20000

HUACtrl

HUACtrl

(b)






1000

600

800

400

0

200

0 4 8

(week)

Urin

ary

albu

min

(120583g

day)

HUACtrl

lowastlowast

lowast

(a)

(kDa)

10

15

20

25

37

50

75

100

150

250

MWUrineSerum

(b)

HUACtrl

(c)

HUA

HUA

Ctrl0

10

5

15

20

Posit

ive a

rea

glom

erul

us (

)

lowastlowast

Ctrl

(d)

Figure 3 Continued


HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)









Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1000

600

800

400

0

200

0 4 8

(week)

Urin

ary

albu

min

(120583g

day)

HUACtrl

lowastlowast

lowast

(a)

(kDa)

10

15

20

25

37

50

75

100

150

250

MWUrineSerum

(b)

HUACtrl

(c)

HUA

HUA

Ctrl0

10

5

15

20

Posit

ive a

rea

glom

erul

us (

)

lowastlowast

Ctrl

(d)

Figure 3 Continued


HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)









Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HUACtrl

HUACtrl0

10

20

30

40

50

Posit

ive a

rea

glom

erul

us (

)

lowast

(e)









Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctrl

(a)

HUA

(b)

MV

MV

HUA

(c)






HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HUACtrl0

500

1000

1500

2000 lowastlowastU

rinar

y 8O

HdG

(ng

day)

(a)

HUACtrl

(b)

HUACtrl0

1

2

3 lowast

Posit

ive a

rea

glom

erul

i (

)

(c)






00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

00 05 10 2015

Urin

ary

albu

min

Cr

Urinary 8OHdGCr

R2 = 049

P lt 001

HUA

Ctrl

(a)

00

05

10

15

Urin

ary

albu

min

Cr

P lt 001

R2 = 029

HUA

Ctrl

110 120 130 140 150

Blood pressure

(b)

00 05 10 15

0

5

10

15

Des

min

pos

itive

area

Urinary 8OHdGCr

P lt 001

R2 = 080

HUA

Ctrl

(c)

00 05 10

0

100

200

300

400

Urinary 8OHdGCr

R2 = 043

HUA

Ctrl

120572-S

MA

pos

itive

area

P lt 005

(d)



HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HUA

Systo

lic b

lood

pre

ssur

e (m

mH

g)

110

120

130

140

150 lowast

HUA+ TEMP

(a)120572

-SM

A p

ositi

ve ar

ea (120583

m2)

0

100

200

300

400lowast

HUA HUA+ TEMP

(b)

0

500

1000

1500

2500

2000

Urin

ary

albu

min

(120583g

day)

NS

HUA HUA+ TEMP

(c)






4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4000

3000

2000

1000

0Ctrl HUA

P = 0071Xa

nthi

ne o

xido

redu

ctas

e act

ivity

(pm

olm

inm

g pr

otei

n)



4 Conclusion


Disclosure


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















podocyte injury and albuminuria in experimental hyperuricemic...

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