Research Article Renal Oxidative Stress Induced by Long-Term Hyperuricemia Alters Mitochondrial Function and Maintains Systemic Hypertension

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1 Oxidtive Medicine nd Cellulr Longevity Volume 15, Article ID , 8 pges Reserch Article Renl Oxidtive Stress Induced y Long-Term Hyperuricemi Alters Mitochondril Function nd Mintins Systemic Hypertension Mgdlen Cristól-Grcí, 1 Fernndo E. Grcí-Arroyo, 1, Edili Tpi, 1, Horcio Osorio, 1, Arhm S. Arellno-Buendí, 1, Mgdlen Mdero, 1 Bernrdo Rodríguez-Iture, 3 José Pedrz-Chverrí, Frncisco Corre, 5 Cecili Zzuet, 5 Richrd J. Johnson, 6 nd Lur-Griel Sánchez Lozd 1, 1 Deprtment of Nephrology, INC Igncio Chávez, 18 Mexico City, DF, Mexico Lortory of Renl Physiopthology, INC Igncio Chávez, 18 Mexico City, DF, Mexico 3 Division of Nephrology, Hospitl Universitrio de Mrcio nd Lortory of Immunoiology, Instituto Venezolno Investigciones Científics, Mrcio 11, Zuli, Venezuel Deprtment of Biology, Fcultd de QuímicUNAM,51MexicoCity,DF,Mexico 5 Deprtment of Crdiovsculr Biomedicine, INC Igncio Chávez, 18 Mexico City, DF, Mexico 6 Division of Renl Diseses nd Hypertension, University of Colordo, Denver, CO 85, USA Correspondence should e ddressed to Lur-Griel Sánchez Lozd; lgsnchezlozd@gmil.com Received 9 Decemer 1; Revised 6 Mrch 15; Accepted 7 Mrch 15 AcdemicEditor:SwrnJ.S.Flor Copyright 15 Mgdlen Cristól-Grcí et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. We ddressed if oxidtive stress in the renl cortex plys role in the induction of hypertension nd mitochondril ltertions in hyperuricemi. A second ojective ws to evlute whether the long-term tretment with the ntioxidnt prevents renl oxidtive stress, mitochondril ltertions, nd systemic hypertension in this model. Long-term (11-1 weeks) nd short-term (3 weeks) effects of oxonic cid induced hyperuricemi were studied in rts (OA, 75 mg/kg BW), OA+Allopurinol (AP, 15 mg/l drinking wter), OA+ (T, 15 mg/kg BW), or vehicle. Systolic lood pressure, renl lood flow, nd vsculr resistnce were mesured. Tuulr dmge (urine N-cetyl-β-D-glucosminidse) nd oxidtive stress mrkers (lipid nd protein oxidtion) long with ATP levels were determined in kidney tissue. Oxygen consumption, conitse ctivity, nd uric cid were evluted in isolted mitochondri from renl cortex. Short-term hyperuricemi resulted in hypertension without demonstrle renl oxidtive stress or mitochondril dysfunction. Long-term hyperuricemi induced hypertension, renl vsoconstriction, tuulr dmge, renl cortex oxidtive stress, nd mitochondril dysfunction nd decresed ATP levels. Tretments with nd llopurinol prevented these ltertions. Renl oxidtive stress induced y hyperuricemi promoted mitochondril functionl disturnces nd decresed ATP content, which represent n dditionl pthogenic mechnism induced y chronic hyperuricemi. Hyperuricemi-relted hypertension occurs efore these chnges re evident. 1. Introduction Over recent yers, epidemiologicl studies nd clinicl intervention trils, including rndomized controlled trils, hve shown tht hyperuricemi is likely cuse or excerting fctor of hypertension nd progressive kidney disese [1 3]. Experimentl studies demonstrted tht experimentl hyperuricemic hypertension is ssocited with inflmmtion, renl microvsculr dmge, nd renl vsoconstriction [ 8]. Oxidtive stress seems to e primry deletereous effect induced y incresed uric cid (UA) []. In this regrd,

2 Oxidtive Medicine nd Cellulr Longevity theroleofthectivtionofnadphoxidseyhyperuricemi hs een well estlished [, 9, 1]. On the other hnd,lrgefrctionofrectiveoxygenspecies(ros)cn e of mitochondril origin, nd mitochondril normlities hve lso een descried during hypertension [11, 1]. We previously reported tht UA induced reduction of mitochondril mss with concomitnt depletion of conitse (ACO- ) nd enoyl CoA hydrtse-1 (ECoAH-1) in endothelil cells [13]. In ddition, hyperuricemic rts hd lower mitochondril DNA (mtdna) in the renl cortex in ssocition with higher levels of intrrenl UA nd oxidtive stress [13]. Since long-term regultion of lood pressure is regulted y kidney, it is importnt to estlish whether hyperuricemi induced hypertension my e secondry effect of incresed renl oxidtive stress. Therefore, the first ojective of this study ws to ddress if oxidtive stress in the renl cortex might ply role in the induction of hypertension nd mitochondril ltertions in this model. A second ojective ws to evlute whether the long-term tretment with the ntioxidnt prevents renl oxidtive stress, mitochondril ltertions, nd systemic hypertension.. Methods All experiments were performed in mle Sprgue-Dwley rts in ccordnce with the Mexicn Federl Regultion for Animl Experimenttion nd Cre (NOM-6-ZOO- 1) nd were pproved y Bioethics nd Investigtion Committees of Instituto Ncionl de Crdiologí Igncio Chvez..1. Short-Term Study. In order to determine whether oxidtive stress plys role in the development of hyperuricemic hypertension nd mitochondril ltertions studies were done in two groups of rts (n = ech) tht received the inhiitor of uricse oxonic cid (OA, 75 mg/kg BW, orl) or vehicle until they developed systemic hypertension (three weeks). In these nimls, lood pressure nd mitochondril function, UA content nd oxidtive stress were evluted in the renl cortex... Long-Term Study. In order to evlute the long-term effect of ntioxidnt therpy on systemic hypertension, renl function, nd mitochondril respirtory cpcity chnges induced y hyperuricemi, the following groups of rts were studied (n =1ech): OA (75mg/kg BW), OA+ (T, 15 mg/kg BW orl), or vehicle for 11-1 weeks. In ddition we compred the effect of preventing the rise of UA induced y OA y dosing llopurinol concomitntly in one dditionl group (AP, 15 mg/l drinking wter). At the end of the study, seven rts were scrificed with pentoritl; the kidneys were excised nd the cortex nd medull surgiclly seprted. The left kidney corticl tissue ws used to isolte mitochondri, nd the right kidney cortex ws snp frozen for dditionl studies. Five rts from ech group were used to determine renl lood flow nd vsculr resistnce. 3. Mesurements Systolic lood pressure (SBP) ws mesured in conscious rts y vlidted volume-sed til cuff method [1]. Plsm UA ws mesured using commercil kit (DCL Dignostics, Chrlottetown, Cnd) Renl Blood Flow. Rts were nesthetized with sodium pentoritl (3 mg/kg, i.p.) nd plced on homeothermic tle. Rts were mintined under euvolemi y infusion of isotonic ovine serum lumin (BSA, 5 mg/dl) during surgery, followed y n infusion of physiologic sline (.9%). Men rteril pressure (MAP) ws continuously monitored. An ultrsound flow proe (TS, Trnsonic Systems, Ithc, NY, USA) ws plced round the left renl rtery to record renl lood flow (RBF). Renl vsculr resistnce (RVR) nd renl plsm flow (RPF) were clculted ccordingly to the formuls RVR = MAP/RBF nd RPF = RBF (1 Hct), respectively. 3.. Mitochondril Studies. Mitochondri were isolted from the renl cortex y differentil centrifugtion s previously descried [15]. Proteins were mesured y the Brdford method. Mitochondril oxygen consumption ws mesured using Clrk-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH, USA). Stte respirtion rte ws evlutedin1.5mlofsicmediumcontining15mmkcl, 1 mm HEPES, 3 mm Pi nd 1 mm succinte plus 1 μg/ml rotenone, or 5 mm sodium glutmte plus 5 mm sodium mlte. Stte 3 respirtion rte ws mesured fter ddition of μm ADP. The respirtory control index (RC) ws clculted s the rtio etween stte 3/stte rtes. Aconitse ctivity ws mesured in isolted mitochondri s the formtion of cis-conitte from isocitrte t nm in Tris-HCl uffer, ph 7. in medium contining isocitrte nd MnCl. One unit ws defined s the mount of enzyme necessry to produce 1 μmol of cis-conitte/min. UA ws extrcted from mitochondri nd mesured using commercil kit (DCL Dignostics, Chrlottetown, Cnd). Vlues of UA were normlized y protein concentrtion Oxidtive Stress. Tissue ws homogenized in phosphte uffer contining cocktil of proteses inhiitors. Protein cronyls nd -hydroxynonenl (-HNE) were mesured using previously pulished methods [16]. 3.. ATP Content. Renl corticl ATP levels were mesured y ioluminescence using commercil kit (ATP Bioluminescence Assy Kit CLS II, Roche Moleculr Biochemicls, Mnnheim, Germny) ccordingly to the mnufcturer instructions Sttisticl Anlysis. Vlues re expressed s men ± stndrd devition (SD). For long-term study, significnt differences etween the three groups were determined y two-wy ANOVA. When the ANOVA P vlue ws <.5,

3 Oxidtive Medicine nd Cellulr Longevity 3 Tle 1: Functionl nd mitochondri dt in long-term follow-up groups. Prmeter Vehicle OA OA + OA + AP Plsm UA (μmol/l) ± 11± ± 36 6 ± 6 Systolic lood pressure (mmhg) 15 ± 1± 7 11 ± 5 19 ± 8 RPF (ml/min) 3.7 ±.3.7 ±.. ±.3.1 ±.3 RVR (mmhg/ml/min) 19 ± 1 8± 5 15 ± 1 17 ± 3 Mitochondril respirtion mlte/glutmte Stte 3 (ng AtO /min/mg prot) 83 ± 18 3 ± ± 11 ± 3 Stte (ng AtO /min/mg prot) 3 ± 6 c 3 ± 11 c 57 ± 1 37 ± 5 c Mitochondril respirtion succinte/rotenone Stte 3 (ng AtO /min/mg prot) 17 ± 6 8 ± ± ± 6 Stte (ng AtO /min/mg prot) 7 ± 53 ± 5 87 ± 65 ± 1 Dt re expressed s men ± SD. RPF = renl plsm flow; RVR = renl vsculr resistnce. P<.5 versus control; P<.5 versus OA; c P<.5 versus. posttest comprisons were mde using Bonferroni multiplecomprison test. For the short-term studies, significnt differences etween OA nd control groups were determined y Student s t-test. Correltion nlysis ssessed the reltionship etween vriles. Sttisticl nlysis ws performed with Prism version 5. (Grph Pd Softwre, Sn Diego, CA, USA).. Results.1. Short-Term Hyperuricemi Induced Systemic Hypertension ut Not Renl Oxidtive Stress Neither Mitochondril Altertions (Figure 1). OA-induced hyperuricemi ws ssocited with the development of systemic hypertension fter three weeks of OA dosing (Figure 1). At this time point, mitochondri from these nimls showed incresed sl respirtion rte (stte ) with oth NADH linked sustrtes nd with succinte. Stte 3 respirtory rte ws lso incresed, indicting oxidtive phosphoryltion coupling irrespective of OA tretment (Figure 1).Theseresultswereingreement with similr ctivity of mitochondril conitse nd equivlent UA concentrtions in the control nd OA-treted group (Figure 1). No chnges in oxidtive stress mrkers in the renl cortex were oserved in the short-term studies (Figure 1)... Long-Term Hyperuricemi Induced Hypertension, s well s Renl Hemodynmic nd Mitochondril Anormlities: Antioxidnt Tretment Successfully Prevented Those Altertions (Figure nd Tle 1)..1. Systemic nd Renl Hemodynmics. Long-term OA tretment induced hyperuricemi, hypertension, decresed renl lood nd plsm flow, nd incresed renl vsculr resistnce. Antioxidnt tretment with did not preventtheriseinplsmua;however,itlockedthedevelopment of systemic hypertension s well s renl hemodynmic chnges (Figure nd Tle 1). Allopurinol tretment prevented hyperuricemi, the ssocited hypertension s well s the renl hemodynmic chnges, s previously reported (Tle 1 nd Figure 1)[7, 8].... Mitochondril Studies. Mitochondril respirtory rte coupled to ATP production ws depressed in the renl cortex of OA-treted hyperuricemic rts. Tretment with oth nd llopurinol mintined mitochondril respirtion similr to control nimls. In ddition, the ctivity of conitse, mrker of superoxide rdicl dmge [17], ws significntly reduced in hyperuricemic rts nd ws preserved in norml levels in the nd llopurinol treted rts. Finlly, mitochondril UA ccumultion ws induced y OA tretment; this effect ws fully prevented y nd llopurinol tretments (Tle 1 nd Figure ). Mitochondril UA ws negtively correlted with conitse ctivity (P <.1)...3. Mrkers of Oxidtive Stress nd ATP Levels in the Renl Cortex. OA-treted rts hd incresed lipid peroxidtion nd cronyl protein oxidtion products. nd llopurinol tretments prevented the oxidtive stress induced y hyperuricemi. Accordingly, ATP content ws reduced in the renl cortex of hyperuricemic nimls. nd llopurinol tretments preserved the concentrtion of renl corticl tissue ATP in similr vlues s control rts. 5. Discussion Long-term hypertension is defect intrictely ssocited with kidney functionl nd/or structurl ltertions. On the other hnd, hyperuricemi hs een descried s risk fctor for the development of systemic hypertension nd chronic kidney disese [1 3]. A primry mechnism involved in those uric cid-medited effects is significnt increment in oxidtive stress [, 13]. Another potentil, s well s poorly explored mechnism of dmge induced y hyperuricemi, is n norml mitochondril function. Therefore, these studies were designed to shed more light on the prticiption of renl oxidtive stress s cusl mechnism to induce hyperuricemic hypertension. In ddition, we evluted the tretment with n ntioxidnt in long-term hyperuricemi nd performed functionl studies in renl cortex isolted

4 Oxidtive Medicine nd Cellulr Longevity 15 Plsm uric cid P <.1 1. Renl cortex oxidized proteins (μmol/l) 1 5 (nmol/mg prot) Control OA Control OA Systolic lood pressure 16 P <.1.8 Renl cortex lipoperoxidtion (mmhg) (nmol/mg prot).6.. Stte 3/stte Control OA Control OA Mlte/glutmte respirtory control rte Mitochondril function (nmol cis-conitte/mg prot) Aconitse ctivity Control OA Control OA 6 Stte 3/stte Succinte/rotenone 1 Mitochondril UA respirtory control rte 3 1 (μg/mg prot) 8 6 Control OA Control OA Figure 1: Short-term hyperuricemi induced systemic hypertension ut not renl oxidtive stress nd mitochondril ltertions. Three weeks of oxonic cid dosing induced hyperuricemi nd systemic hypertension, ut not renl oxidtive stress neither renl mitochondril functionl normlities. mitochondri in order to define its potentil role s n dditionl injurious effect of hyperuricemi. Three weeks were required for hyperuricemi to induce hypertension in rts. At this time point, we did not find evidence of incresed renl corticl oxidtive stress in hyperuricemic nimls. Thus, these dt suggest tht the estlishment of systemic hypertension during hyperuricemi is n event independent of renl oxidtive stress. The mechnisms

5 Oxidtive Medicine nd Cellulr Longevity 5 5 Renl cortex oxidized proteins 1. Renl cortex lipoperoxidtion DNPH (nmol/mg prot) 3 1 -HNE (nmol/mg prot) Mitochondril function Stte 3/stte 5 3 Mlte-glutmte respirtory control rte (μg/mg prot) 3 1 Mitochondril UA 1 Stte 3/stte Succinte-rotenone respirtory control rte (nmol cis-conitte/mg/min) 6 Aconitse ctivity (nnom ATP/mg tissue) ATP content in renl cortex Figure : Antioxidnt tretment successfully prevented long-term hyperuricemi induced renl iochemicl nd mitochondril normlities. Long-term OA-induced incresed oxidtive stress nd mitochondril ccumultion of UA. These chnges were ssocited with renl corticl mitochondril dysfunction chrcterized y oxidtive phosphoryltion uncoupling (decresed RC) nd decresed conitse ctivity nd diminished ATP renl content. nd llopurinol tretments prevented those chnges. : P <.5 versus Control; : P <.5 versus OA.

6 6 Oxidtive Medicine nd Cellulr Longevity involved during this initil phse re likely relted to decresed systemic nitric oxide iovilility [5]. In short-term hyperuricemi, mitochondril oxygen consumption, conitse ctivity, nd uric cid were not different etween renl cortex mitochondri isolted from oxonic cid treted nimls nd those isolted from control group. Norml renl cortex mitochondri cutely exposed to uric cid showed ltertions neither in mitochondril respirtion nor in conitse ctivity (dt not shown). Therefore, these dt suggest tht mitochondril functionl ltertions re not n erly mnifesttion of hyperuricemi pthogenesis in kidney. Nevertheless, some mitochondril functionl normlities induced y uric cid hve een descried in endothelil cultured cells [18]. Previously we showed incresed mtdna dmge nd oxidtive stress induced y long-term hyperuricemi in renl corticl tissue [8]. However, it is not known whether mitochondril functionl sttus is ltered in the kidney of long-term hyperuricemic rts, nor if such effects cn e modulted y oxidtive stress. Therefore, second ojective of these studies ws to evlute whether the tretment with the ntioxidnt cn provide enefit on mitochondril oxygen consumption nd conitse ctivity fter long-term hyperuricemi, s ll of these functions re prticulrly sensitive to rective oxygen species induced injury [1]. We lso compred effects with those provided y llopurinol, drug tht locks the development of hyperuricemi induced y oxonic cid in rts, s well s the hypertension nd renl ltertions relted to incresed uric cid [8, 19]. In the present studies, we oserved tht nd llopurinol provided similr therpeutic enefits in longterm hyperuricemi (11-1 weeks); llopurinol effects likely were relted to its ntihyperuricemic effect [7, 8]; in contrst, did not lock oxonic cid-induced hyperuricemi ut prevented the increment in lood pressure, intrrenl oxidtive stress, nd the renl hemodynmic ltertions ssocited with incresed levels of uric cid. Previously we reported similr effects of in nimls exposed to the effects of hyperuricemi for shorter term (5 weeks) []. On the other hnd, the mintennce of intrrenl ntioxidnt systems is fundmentl to preserve kidney function nd structure. In this regrd, physiologicl concentrtions of renl dopmine hve protective effects on oxidtive stress in the kidney. Moreover, dopmine cting on its receptors D1R, DR, nd D5R inhiits NADPH oxidse ctivity nd ROS productionndlsostimultesntioxidntenzymessuchs SOD, glutthione peroxidse, glutmyl cysteine trnsferse, nd HO-1, mong others []. It is unknown whether uric cid lters renl dopmine system; however, there is some evidence of n interction etween them [1]. After long-term hyperuricemi, renl mitochondril functionl ltertions were noticed; sl respirtion rte (stte ) ws not different mong the groups with NADH linked sustrtes (mlte/glutmte) or with succinte. However, respirtion coupled to ATP production (stte 3) ws significntly reduced in renl corticl mitochondri of OAtreted nimls with either mlte/glutmte or succinte. Therefore, reduced vlues of respirtory control rtes were indictive of oxidtive phosphoryltion uncoupling. nd llopurinol tretments prevented mitochondril dmge, s respirtory control rtes were preserved with vlues similr to control rts with mlte/glutmte nd with succinte. Mitochondril conitse ctivity ws lso evluted, s its inctivtion is mrker of superoxide-medited mitochondril dmge. Aconitse ctivity in OA-treted rts ws significntly lower thn tht in control, OA+AP nd OA+. In ddition, we found tht mitochondril lystes from OA-treted nimls hd significntly higher uric cid, nd this effect ws prevented with llopurinol tretment. Interestingly lso prevented mitochondril uric cid ccumultion. Mitochondril uric cid results from t lest three mechnisms: mitochondril xnthine oxidse ctivity [], mitochondril purine ctolism [3],nduptkefrom cytoplsmic spce []. Regultion of systemic nd intrcellulr uric cid levels is complex process tht requires the prticiption of severl different trnsporters for its uptke nd its efflux. Therefore, dysregultion of such equilirium my lter intr- nd extrcellulr concentrtions of UA. Plnt-derived sustnces with ntioxidnt ctivity such s quercetin [5] nd nuciferine [6] prevented hyperuricemi induced y oxonic cid in mice y ltering the expression nd ctivity of vrious uric cid trnsporters. It hs een shown tht, in order to exert deleterious effects, UA depends on its intrcellulr concentrtion. In this context, the regultion of UA trnsporters is of utmost importnce. Verzol et l. showed tht URAT1 is fundmentl for UA to enter into proximl tuulr cells nd induce intrcellulr oxidtive stress [7]. In the present studies, we found tht tretment hd eneficil effect despite the fct tht it did not reduce UA plsm levels. These results suggest tht might lso differentilly regulte UA trnsporters, overll resulting in decresed levels of intrcellulr UA, thus conferring protection. In support to this contention, we found tht ws le to prevent intrmitochondril increment of UA. Since we found diminished respirtion coupled to ATP production with long-term oxonic cid-induced hyperuricemi, we lso quntified ATP concentrtion in corticl tissue of the vrious groups. Chronic hyperuricemi induced significnt reduction of ATP content in renl cortex, which ws similr to our previous findings in endothelil cells [13]. This effect ws prevented y the tretment with nd prtilly with llopurinol, therefore suggesting tht preservtion of mitochondril respirtion coupling my contriute to mintining ATP renl content. We hve previously shown tht chronic hyperuricemi induces glomerulr hypertension nd renl corticl vsoconstriction [8, 19]. In the present studies, we directly mesured renl lood flow nd confirmed reduction in this prmeter [8, 19]. nd llopurinol tretments successfully prevented ll these renl functionl ltertions. In summry, in the estlishment of hyperuricemic hypertension, there is dissocition of renl oxidtive stress nd hypertension tht likely reflects n intct intrrenl ntioxidnt system t tht erly time point. On the other hnd, during long-term hyperuricemi, renl oxidtive stress is key mechnism for sustining systemic hypertension,

7 Oxidtive Medicine nd Cellulr Longevity 7 which is physiologic response to persistent renl vsoconstriction. In ddition, the present studies suggest tht renl oxidtive stress induced y hyperuricemi likely promotes mitochondril functionl disturnces nd decresed ATP content, which represent n dditionl pthogenic mechnism induced y chronic hyperuricemi. Conflict of Interests Richrd J. Johnson is listed s n inventor on ptents nd ptent pplictions relted to lowering uric cid in metolic nd renl disese. Richrd J. Johnson is lso on the Scientific Bord of Amwy nd XORT Therpeutics, the ltter which is developing xnthine oxidse inhiitors for the tretment of dolescent hypertension. Acknowledgments Funding ws supported y Ntionl Council of Science nd Technology (CONACyT) Mexico Grnts nos. 1333, 16799, 17757, nd References [1] P. C. Gryson, S. Y. Kim, M. Lvlley, nd H. K. Choi, Hyperuricemi nd incident hypertension: systemtic review nd met-nlysis, Arthritis Cre & Reserch,vol.63,no.1,pp. 1 11, 11. 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9 MEDIATORS of INFLAMMATION The Scientific World Journl Gstroenterology Reserch nd Prctice Journl of Dietes Reserch Interntionl Journl of Journl of Endocrinology Immunology Reserch Disese Mrkers Sumit your mnuscripts t BioMed Reserch Interntionl PPAR Reserch Journl of Oesity Journl of Ophthlmology Evidence-Bsed Complementry nd Alterntive Medicine Stem Cells Interntionl Journl of Oncology Prkinson s Disese Computtionl nd Mthemticl Methods in Medicine AIDS Behviourl Neurology Reserch nd Tretment Oxidtive Medicine nd Cellulr Longevity