Oxidative stress implies a shift in the balance between the

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1 Superoxide Dismutse 1 Limits Renl Mirovsulr Remodeling nd Attenutes Arteriole nd Blood Pressure Responses to Angiotensin II vi Modultion of Nitri Oxide Biovilility Mttis Crlström, En Yin Li, Zufu M, Andres Steege, Andres Ptzk, Ulf J. Eriksson, Jon O. Lunderg, Christopher S. Wilox, A. Erik G. Persson Downloded from y on Otoer 7, 218 Astrt Oxidtive stress is ssoited with vsulr remodeling nd inresed preglomerulr resistne tht re oth implited in the pthogenesis of renl nd rdiovsulr disese. Angiotensin II indues superoxide prodution, whih is metolized y superoxide dismutse (SOD) or svenged y NO. We investigted the hypothesis tht SOD1 regultes renl mirovsulr remodeling, lood pressure, nd rteriolr responsiveness nd sensitivity to ngiotensin II using SOD1-trnsgeni () nd SOD1-knokout () mie. Blood pressure, mesured telemetrilly, rose more ruptly during prolonged ngiotensin II infusion in mie. The fferent rteriole medi:lumen rtios were redued in nd inresed in mie. Afferent rterioles from nontreted wild types hd grded ontrtion to ngiotensin II (sensitivity: 1 9 mol/l; responsiveness: 4%). Angiotensin II ontrtions were less sensitive (1 8 mol/l) nd responsive (14%) in ut more sensitive (1 13 mol/l) nd responsive (89%) in mie. Arterioles from hd 4-fold inresed superoxide formtion with ngiotensin II t 1 9 mol/l. N G -nitro-l-rginine methyl ester redued rteriole dimeter of nd enhned ngiotensin II sensitivity nd responsiveness of nd mie to the level of mie. SOD mimeti tretment with Tempol inresed rteriole dimeter nd normlized the enhned sensitivity nd responsiveness to ngiotensin II of mie ut did not ffet or mie. Neither SOD1 defiieny nor overexpression ws ssoited with hnges in nitrte/nitrite exretion or renl mrna expression of NO synthse, NADPH oxidse, or SOD2/SOD3 isoforms nd ngiotensin II reeptors. In onlusion, SOD1 limits fferent rteriole remodeling nd redues sensitivity nd responsiveness to ngiotensin II y reduing superoxide nd mintining NO iovilility. This my prevent n erly nd exggerted lood pressure response to ngiotensin II. (Hypertension. 21;56: ) Key Words: fferent rterioles CuZnSOD hypertension ICSOD oxidtive stress superoxide SOD1 kidney Oxidtive stress implies shift in the lne etween the prodution of retive oxygen speies (ROS) nd the tion of ntioxidnt systems nd hs een implited in the pthogenesis of renl nd rdiovsulr disese. 1,2 NADPH oxidse is n importnt soure of superoxide (O 2 ) in the vsulture. O 2 tht espes metolism y superoxide dismutse (SOD) n intivte NO, 3 nd growing evidene demonstrtes tht oxidtive stress nd NO defiieny in the kidney ontriute to vsulr dysfuntion nd hypertension. 3 Furthermore, potentil rosstlk etween NADPH oxidse nd SOD tivities hs een suggested. 4,5 SOD exists s 3 different isoforms: opper-zin SOD (SOD1), predomintely loted in the ytoplsm; mngnese SOD (SOD2) in the mitohondri; nd extrellulr SOD (SOD3) in the extrellulr spe. 6 SOD1 ounts for 6% to 8% of SOD tivity in the kidneys nd lso hs n importnt role in lood vessels, where it preserves NO relese from the endothelium. 7,8 The renl fferent rterioles re the mjor resistne vessels to the kidneys nd ply n importnt role in lood pressure regultion. 9 The fferent rteriolr tone nd resistne re determined y the lne etween onstritor gents, suh s ngiotensin II (Ang II) nd vsodiltor pthwys, notly NO. 1 Reeived July 9, 21; first deision August 4, 21; revision epted August 31, 21. From the Deprtment of Medil Cell Biology (M.C., E.Y.L., Z.M., U.J.E., A.E.G.P.), Uppsl University, Uppsl, Sweden; Deprtment of Mediine (M.C., E.Y.L., C.S.W.), Georgetown University Medil Center, Wshington, DC; Deprtment of Physiology nd Phrmology (M.C., J.O.L.), Krolinsk Institute, Stokholm, Sweden; Deprtment of Nephrology (Z.M.), Tongji Hospitl, Tongji Medil College, Huzhong University of Siene nd Tehnology, Wuhn, People s Repuli of Chin; Institute of Vegettive Physiology (A.P.), Chrité-Universitätsmedizin Berlin, Berlin, Germny; Internl Mediin II/Nephrology (A.S.), University Medil Center Regensurg, Regensurg, Germny. M.C., E.Y.L., Z.M., C.S.W., nd A.E.G.P. ontriuted eqully to this work. Correspondene to Mttis Crlström, Deprtment of Mediine, Georgetown University, Building D-38, 4 Reservoir Rd, Wshington, DC 257. E-mil mttis.rlstrom@m.uu.se 21 Amerin Hert Assoition, In. Hypertension is ville t DOI: /HYPERTENSIONAHA

2 98 Hypertension Novemer 21 Downloded from y on Otoer 7, 218 Infusions of Ang II indue NO relese in fferent rterioles, 11 ut the response to this is limited y onomitnt inrese in O 2 produed y NADPH oxidse. 8,12 Indeed, n infusion of Ang II my produe suffiient ROS to overwhelm the ntioxidnt systems, with susequent NO defiieny, hyperretivity, nd remodeling of fferent rterioles. 13,14 Inresed rteriolr retivity my redue renl perfusion nd filtrtion, inrese extrellulr volume, nd, thus, ontriute to the development of Ang II indued hypertension. 15,16 These properties of ROS undersore the importne of systems tht limit O 2 in djusting renl rteriolr tone. However, there is less informtion regrding the funtionl signifine of seleted SOD isoforms in the regultion of renl miroirultion nd lood pressure. We tested the hypothesis tht SOD1 regultes renl mirovsulr remodeling nd limits fferent rteriolr responsiveness nd hypertension in response to Ang II. Mterils nd Methods Experimentl Design The experiments were onduted on homozygous littermtes from heterozygous reeding pirs of SOD1-trnsgeni (; C57BL/ 6-Tg[SOD1]3Cje/J [stok No. 2629]) or SOD1-knokout (SOD1- ko; B6;129S7-Sod1tm1Le/J [stok No. 2972]) mie from the Jkson Lortory (Br Hror, ME). Wild-type littermtes from the reeding olonies served s ontrols. Genotyping of the offspring were performed s desried previously (plese see the online Dt Supplement t 17 Both sexes were used, with equl distriution nd similr ge (1 to 16 weeks) in every set of experiments. The nimls were fed stndrdized mouse how (.7% NCl, R36, th SD389, Ltmin) nd tp wter d liitum. Telemetri lood pressure mesurements were performed in wildtype nd mie efore nd fter suutneous infusion of slow-pressor dose of Ang II for 14 dys. Renl exretion of nitrte/nitrite ws nlyzed to determine totl NO prodution. All of the fferent rteriole experiments (ie, isotoni ontrtions nd hnges in O 2 levels) nd gene expression studies were performed in nontreted mie (ie, without prolonged Ang II infusion). Blood Pressure Response to Prolonged Ang II Infusion Telemetri devies (PA-C1, DSI) were implnted in nd mie. After surgery, the nimls were llowed to reover for 1 dys. Therefter, the lood pressure ws mesured ontinuously for 96 hours to determine sl levels (plese see the online Dt Supplement). The mie were nesthetized y spontneous inhltion of isoflurne (Forene, Aot Sndinvi AB) in ir ( 2.2%), nd n osmoti minipump (ALZET, Duret) ws implnted suutneously, delivering Ang II (Sigm-Aldrih) t 4 g/kg per 24 hours for 14 dys. After implnttion, lood pressure ws reorded ontinuously throughout the Ang II delivery period. Renl Exretion of Nitrte/Nitrite Mie were pled in metoli ges for 24 hours, with food nd wter given d liitum. Wter onsumption nd urine prodution were mesured grvimetrilly. Smples of fresh urine were stored t 7 C until nlysis. Nitrte nd nitrite in urine were mesured with dedited high-performne liquid hromtogrphy system (ENO-2, EiCom) desried previously (plese see the online Dt Supplement). 18 Afferent Arteriole Mesurements Dissetion nd Perfusion of Arterioles Dissetion nd perfusion were performed s desried previously (plese see the online Dt Supplement). Figure 1. Setup for the renl fferent rteriole experiments. The mirophotogrph shows glomerulus nd its fferent rteriole held y 2 holding pipettes. The perfusion pipette (5- m dimeter), inserted into the holding pipette on the right side, ws onneted to reservoir ontining the perfusion solution to provide pressure of 1 mm Hg in the pressure hed, whih produed flow of 5 nl/min. The inner luminl dimeter (Ø) of the rteriole ws mesured t the most tive site to estimte the effet of vsotive sustnes. Mesurements of Arteriolr Dimeter The tehnique for the renl fferent rterioles is demonstrted in Figure 1. The experiments were reorded y video system, digitized offline, nd nlyzed s desried previously. 11 The inner luminl dimeters of the rterioles were mesured t the most tive site to estimte the effet of vsotive sustnes. Responsiveness (ie, mximl hnge in rteriolr dimeter) nd sensitivity (ie, threshold onentrtion of Ang II mediting signifint hnge in rteriole dimeter) were lulted to investigte the roles of NO nd O 2 for the Ang II indued ontrtion. In ll of the series, the lst 1 seonds of ontrol or tretment period were used for sttistil nlysis of stedy stte responses. Eh experiment used seprte disseted fferent rteriole: only 1 rteriole ws used per niml. The inner luminl dimeter nd medi thikness were mesured during seline (efore pplition of ny sustnes), nd the res were lulted to ompute the medi:lumen rtios to ssess vsulr remodeling. O 2 Mesurements in Afferent Arterioles O 2 genertion ws ssessed y fluoresene mirosopy of perfused fferent rterioles with dihydroethidium or tempo-9ac, s desried previously (plese see the online Dt Supplement). 19 Anlysis of mrna Expression Infusion of old PBS ws strted one the ven v ws ut to remove the lood. The hert nd kidneys were explnted, lotted, nd weighed. The renl ortex ws seprted nd homogenized, nd quntittive PCR nlysis ws performed (plese see the online Dt Supplement). Drugs nd Regents All of the drugs were pplied to the th solution in the rteriolr ontrtion experiments (plese see the online Dt Supplement). Sttistil Anlysis Vlues re presented s men SEM. Repeted-mesures ANOVA ws used to test time- or onentrtion-dependent hnges in the rteriolr dimeter nd to ssess differenes etween the groups. Post ho omprisons were performed with Fisher test. ANOVA, followed y the Fisher post ho test, when pproprite, ws used for nlysis of lood pressure, hnges in O 2 levels, nd vsulr

3 M en Arteril Pressure (mmhg) Men Arteril Pressure (mmhg ) Crlström et l SOD1 Limits Remodeling nd Angiotensin II Responses 99 Tle. Renl nd Crdiovsulr Chrteristis of SOD1 Mie Vrile Wild-Type Kidney nd hert BW, g TKW, g TKW/BW HW, g HW/BW Afferent rterioles Medi re, m Lumen re, m Medi:lumen rtio Afferent rteriole O 2 Chnge with Ang II, % BW indites ody weight; TKW, totl kidney weight; nd HW, hert weight in (n 7), (n 7), nd (n 7) mie. Afferent rterioles inlude evlution of vsulr properties, s mesure for vsulr remodeling, in (n 22), (n 23), nd (n 26) mie. Afferent rteriole O 2 shows hnges in O 2 levels in response to extrluminl pplition of Ang II t 1 9 mol/l in (n 7) nd (n 5) mie. Vlues re presented s men SEM. P.5 ompred with mie. P.5 ompred with mie Bseline Angiotensin II Dys Downloded from y on Otoer 7, 218 remodeling. Wiloxon tests were pplied for gene expression dt. Differenes were onsidered to e sttistilly different if P.5. Ethis The experiments were pproved y the Uppsl Ethil Committee for Animl Experiments nd Georgetown University Animl Cre nd Use Committee nd onduted in ordne with the Ntionl Institutes of Helth Guide for Cre nd Use of Lortory Animls. Results All of the nimls were in good ondition, nd t the time of euthnsi, there were no differenes in ody weights mong the groups. The heterozygous reeding generted lower numer of homozygous offspring ([ /, 29%]; [ /, 52%]; [ /, 19%]), ut no differenes in mendelin distriution were found for the heterozygous reeding. mie displyed higher kidney nd hert msses, djusted for ody weights (Tle). Blood Pressure Response to Prolonged Ang II Infusion There ws no differene in sl lood pressure (ie, 4 dys men) etween the (17 1 mm Hg) nd (11 5 mm Hg) mie. mie displyed drmti initil lood pressure response to Ang II ut rehed similr lood pressure level s mie fter 1 dys of tretment. During the first 4 dys of Ang II infusion, lood pressure inresed with mm Hg in mie (P.5), wheres no signifint hnge ws oserved in mie (3 1 mm Hg). The mie displyed typil slow-pressor response to Ang II with signifint lood pressure response fter 1 dys of tretment. Averged lood pressure dt for seline period nd for the whole Ang II tretment period re presented in Figure 2. Bseline Ang II Bseline Ang II Figure 2. SOD1 prevents rpid nd exggerted lood pressure response to ngiotensin II. Top, Men rteril pressure in onsious (n 5) nd mie (n 5). The telemetri mesurements were onduted ontinuously under ontrol onditions for 4 dys (Bseline) nd then with hroni Ang II infusion t slow-pressor rte (4 g/kg per 24 hours) for 14 dys. Bottom, Men rteril pressure during seline period (4 dys verge) nd the whole Ang II infusion period (14 dys verge). Bsl lood pressures were similr in oth genotypes; however, Ang II elevted lood pressure more in mie thn in mie. Vlues re presented s men SEM. P.5. Renl Nitrte/Nitrite Exretion There were no differenes in renl nitrte/nitrite exretion (in nnomoles per 24 hours per grm of ody weight) mong (37 4; n 7), (5 15; n 7), nd mie (53 2; n 7), inditing tht the systemi NO prodution ws similr in ll 3 of the genotypes. Afferent Arteriole Mesurements To investigte the hypothesis tht inresed rteriolr responsiveness/sensitivity ontriutes to the erly nd rpid lood pressure response oserved in mie, ll of the fferent rteriole experiments were performed in nontreted mie (ie, without prolonged Ang II infusion). Bsl Arteriolr Ares nd Vsulr Remodeling The sl luminl re of fferent rterioles ws signifintly smller in mie thn in nd mie (Tle). No differene ws found etween the nd mie. Compred with mie, the rteriolr medi thikness nd medi:lumen rtios were in-

4 91 Hypertension Novemer 21 Downloded from y on Otoer 7, 218 Luminl Dimeter (%) ontrol Ang II (log mol/l) resed in mie nd redued in mie (Tle). To ontrol for these differenes in seline luminl re, results re presented for solute nd frtionl hnges of the vessels. Plese see the online Dt Supplement for solute hnges in rteriole dimeters. Effet of Ang II Conentrtion-response urves were otined y umultive pplition of Ang II (1 14 to 1 6 mol/l; eh for 2 minutes). Ang II onstrited fferent rterioles from wildtype mie in onentrtion-dependent mnner (threshold response: 1 9 mol/l), with mximum response of 4%. Afferent rterioles of mie were more sensitive (threshold response: 1 13 mol/l) nd more responsive (89%) to Ang II, ut rterioles from mie were less sensitive (threshold response: 1 8 mol/l) nd less responsive (14%) to Ang II (Figure 3). Effet of NO Synthse Inhiition With N G -Nitro-L-Arginine Methyl Ester N G -nitro-l-rginine methyl ester (L-NAME; 1 4 mol/l), pplied for 15 minutes, onstrited fferent rterioles of mie y 8%, ut no ontrtile response ws oserved in the mie. Arterioles from the mie hd stronger ontrtion ( 38%) fter 15 minutes of tretment (Figure 4). Effet of L-NAME Tretment on Ang II Indued Contrtions To investigte the role of NO in offsetting Ang II indued rteriolr ontrtion, vessels were treted with L-NAME (1 4 mol/l) for 15 minutes efore nd during tretment with Ang II (1 14 to 1 6 mol/l; 2 minutes eh dose). L-NAME did not ffet the responsiveness to Ang II in mie ut enhned the response of nd mie to the level of mie (Figure 5). L-NAME tretment inresed the sensitivity to Ang II in (1 13 to 1 14 mol/l) ut to muh higher degree in mie (1 9 to 1 14 mol/l) nd in mie (1 8 to 1 13 mol/l). Figure 3. SOD1 ttenutes renl mirovsulr responses to Ang II. Ang II onentrtion-response urves in isolted nd perfused fferent rterioles of nontreted (n 7), wildtype (n 11), nd (n 7) mie. Vlues re presented s men SEM., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in wildtype mie., P.5 ompred with ontrol period in mie. P.5 ompred with mie. Luminl Dimeter (%) ontrol Time (min) Figure 4. SOD1 is ssoited with inresed NO iovilility in renl mirovessels. Effet of L-NAME (1 4 mol/l) on dimeters of isolted nd perfused fferent rterioles from nontreted (n 7), (n 8), nd (n 8) mie. Vlues re presented s men SEM., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in mie. P.5 ompred with nd mie. Effet of Tempol Tempol (1 4 mol/l) ws pplied for 15 minutes to redue sl levels of O 2. Tempol dilted fferent rterioles of mie y 11% ut did not hnge the dimeter of rterioles from or mie (Figure 6). Effet of Tempol Tretment on Ang II Indued Contrtions To investigte the role of O 2 on Ang II indued rteriolr ontrtion, vessels were treted with Tempol (1 4 mol/l) for 15 minutes efore nd during tretment with Ang II (1 14 to 1 6 mol/l; 2 minutes eh dose). The ddition of Tempol did not influene the responsiveness to Ang II in (18%) or (43%) mie (Figure 7) ut strongly ttenuted the ontrtile response to Ang II in the mie, with redution in the mximl response from 89% to 47%. Tempol tretment did not hnge the sensitivity to Ang II in or Luminl Dimeter (%) ontrol Ang II (log mol/l) Figure 5. During NOS inhiition, the renl mirovsulr responses to Ang II of the different genotypes eme indistinguishle. Effet of L-NAME (1 4 mol/l) on Ang II onentrtion response in isolted nd perfused fferent rterioles of nontreted (n 1), (n 8), nd (n 7) mie. Control period represents the rteriolr dimeter fter L-NAME pretretment for 15 minutes. Vlues re presented s men SEM., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in mie.

5 Crlström et l SOD1 Limits Remodeling nd Angiotensin II Responses 911 Downloded from y on Otoer 7, 218 Luminl Dimeter (%) ontrol Time (min) mie ut normlized the enhned sensitivity in mie (1 13 to 1 9 mol/l). Afferent Arteriolr O 2 Levels Figure 6. SOD mimi tretment diltes renl mirovessels tht lk SOD1. Effet of Tempol (1 4 mol/l) on dimeters of isolted nd perfused fferent rterioles from nontreted (n 6), (n 7), nd (n 7) mie. Vlues re presented s men SEM., P.5 ompred with ontrol period in mie. P.5 ompred with nd mie. Effet of Ang II To investigte whether the enhned ontrtile response to Ang II in mie ws relted to ompromised ntioxidnt defense with inresed oxidtive stress, O 2 levels were mesured in response to Ang II (1 9 mol/l). As shown in the Tle, Ang II indued hnges in O 2 levels were signifintly higher in thn in mie. Gene Expression Studies Rel-time PCR reveled no differenes in renl ortil mrna expression for NO synthse (NOS), NADPH oxidse, or SOD2/SOD3 isoforms or in Ang II reeptors mong the genotypes (plese see the online Dt Supplement). Luminl Dimeter (%) ontrol Ang II (log mol/l) Figure 7. SOD mimi tretment normlizes the exggerted Ang II response of renl mirovessels tht lk SOD1. Effet of Tempol (1 4 mol/l) on Ang II onentrtion response in isolted nd perfused fferent rterioles of nontreted (n 6), (n 7), nd (n 7) mie. Control period represents the rteriolr dimeter fter Tempol pretretment for 15 minutes. Vlues re presented s men SEM., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in mie., P.5 ompred with ontrol period in mie. P.5 ompred with nd mie. Disussion The min new findings re tht the SOD1 ttenutes responsiveness nd sensitivity of renl fferent rterioles to Ang II nd modertes the erly inrese in lood pressure to prolonged Ang II. Furthermore, the study suggests tht SOD1 defiieny is ssoited with renl nd rdi hypertrophy nd hypertrophi remodeling of renl mirovessels. SOD1 is the predominnt isoform in the vsulr wll nd ounts for the mjority of SOD tivity in the kidney. 8,2 Didion et l 21 hve demonstrted tht SOD1 defiieny is ssoited with inresed O 2 levels in rotid rteries nd endothelil dysfuntion, 2 whih n e prevented y SOD mimeti. Furthermore, inresed SOD1 expression mrkedly redued Ang II indued vsulr O 2 levels in the ort. 22 mie re normotensive ut disply renl oxidtive stress nd inresed lood pressure during high sodium tretment, whih n e reversed y tretment with SOD mimeti. 23 An ttenuted lood pressure nd vsulr response to L-NAME hs een reported in SOD3-ko mie 24 nd ws sried to redued NO iotivity. The responsiveness of fferent rterioles to Ang II ws redued in mie ut strongly ugmented in mie. NOS inhiition did not hnge rteriolr dimeter in the mie ut redued the dimeter in mie. This suggests tht the sl NO iovilility in fferent rterioles ws low in mie ut high in mie. This ws onfirmed y the finding tht, fter L-NAME tretment, the ontrtile response to Ang II eme similr mong ll 3 of the genotypes. Furthermore, L-NAME enhned the sensitivity to Ang II in nd mie (1 5 times) to the level of mie. This suggests tht NO iovilility ws ritil determinnt for the differenes in responsiveness nd sensitivity to Ang II mong the groups. mie hve enhned iointivtion of NO nd inresed formtion of peroxynitrite 25 nd lipid peroxidtion. 23 The proposl tht inresed mirovsulr O 2 levels were responsile for the enhned responses to Ang II in mie ws supported y mesurements of Ang II indued hnges in O 2 nd experiments with Tempol in fferent rterioles. Administrtion of Ang II (1 9 mol/l) used 4-fold greter rteriolr ontrtion in mie thn in s, ompnied y 4-fold greter inrese in mirovsulr O 2. Tempol is redox-yling nitroxide tht effetively dismutses O Tempol dilted fferent rterioles in mie nd prevented their exggerted ontrtile response to Ang II. Furthermore, Tempol normlized the enhned sensitivity to Ang II in mie (redued y 1 4 times) ut hd no effets in or mie. This finding is onsistent with previous studies tht the renl vsulr resistne of norml mie is little ffeted y Tempol ut is redued in mie with oxidtive stress euse of prolonged Ang II infusion. 27 Tken together, this demonstrtes tht mie hve inresed mirovsulr oxidtive stress nd redued NO iovilility tht enhnes their response to Ang II. However, the diminished ontrtion to Ang II in mie persisted fter Tempol, inditing the opertion of mehnism other thn redued O 2 tht limited responsiveness in the group.

6 912 Hypertension Novemer 21 Downloded from y on Otoer 7, 218 Oxidtive stress nd NO defiieny hve een implited in mirovsulr remodeling nd endothelil dysfuntion in rdiovsulr disese. 28 Svenging of ROS or overexpression of SOD suppresses Ang II indued hypertrophi remodeling in orti smooth musle ells. 29 Furthermore, endothelil NOS defiient mie nd rts with prolonged NOS lokde displyed vsulr remodeling tht is independent of lood pressure, 28 demonstrting tht sl NO regultes vsulr growth. Thus, renl mirovsulr hypertrophi remodeling in mie nd redued wll thikness of mie my relte to O 2 -dependent hnges in NO iovilility. We hve demonstrted previously tht kidneys from mie displyed hypertrophy nd firoti nd inflmmtory hnges. 23 The present study onfirms renl hypertrophy nd shows further tht SOD1 defiieny used rdi hypertrophy. Our findings tht SOD1 limits vsulr remodeling of fferent rterioles might hve ontriuted to the differenes in ontrtile ehvior. However, our results dissoite the effets of vsulr remodeling from rteriole ontrtile responses to Ang II, euse the responses eme indistinguishle y short-term inhiition of NOS. This indites tht the effets of SOD1 on the response to Ang II re ttriuted to modultion of the offsetting effet of iotive NO. Furthermore, the finding tht Ang II used 4-fold greter inrese in fferent rteriolr O 2 in mie nd tht Tempol normlized the enhned ontrtile responses demonstrtes n importnt role of SOD1 in limiting the vsulr umultion of O 2 during hllenge with Ang II. Evlution of mrna expression in the renl ortex did not revel ny differenes etween nd mie for gene expression of NOS, NADPH oxidse, or SOD2/SOD3 isoforms or Ang II reeptors. Although expressionl studies in fferent rterioles would hve een more onlusive, our renl ortil mesurements indite tht differenes mong the genotypes re minly ttriuted to the ility of SOD1 to metolize O 2 nd modulte NO iovilility rther thn hnges in other key enzymes/reeptors. Studies in SOD3-knokout mie lso hve determined tht lk of this SOD isoform is not ompensted for y other ntioxidtive enzymes. 24 Moreover, unhnged expression of NOS isoforms in the kidney nd similr rtes of nitrte/nitrite exretion mong the genotypes re onsistent with similr overll NO prodution nd suggest tht the mjor effets of SOD1 expression re to inrese NO iovilility. A reent study demonstrted tht glutthione S-trnsferse 4 is ompenstory indued in young mie nd my hve protetive role ginst oxidtive stress. 3 However, the present study suggests tht hnges in glutthione S-trnsferse 4 do not, or only prtly, ompenste for SOD1 defiieny in dult mie. Ang II tretment n tivte NADPH oxidses nd inrese O 2 prodution with susequent renl NO defiieny. The slow-pressor response to Ang II is suggested to e of renl origin, 31 euse the lood pressure is slt sensitive 32 nd is ssoited with elevted renl mirovsulr resistne, 33 enhned Ang II indued responses of fferent rterioles, 12,34 nd slt retention. 35 In the present study, slow-pressor infusion of Ang II ws onverted into pressor infusion in mie in whih it elevted lood pressure within the first dy. Interestingly, SOD3-ko mie lso displyed n exggerted hypertensive response to Ang II t pressor rte, whih ws ttriuted to redued NO iovilility. 8,24,36 In ontrst, Ang II infused t slow-pressor rte led to similr inreses in lood pressure in SOD3-ko nd mie, 8 whih were ttriuted to downregultion of SOD3 nd upregultion of SOD1 in the kidney. 8 Thus, SOD1 ppers to provide mjor defense in the kidney ginst Ang II indued ROS umultion. A limittion of the present study is tht lood pressure responses to Ang II were not investigted in mie. We hve demonstrted previously tht Tempol tretment in mie redued the slow-pressor response to Ang II. 27 Bsed on this finding we would expet mie to hve n ttenuted response ompred with mie given Ang II. The mehnisms responsile for returning lood pressure in mie to levels of mie during prolonged infusion of Ang II remin to e investigted. Our findings provide evidene tht redued ntioxidnt pity, euse of lk of SOD1, rendered the renl mirovsulr system sensitive to hnges in O 2 prodution. Inresed O 2 vilility my ontriute to the exggerted lood pressure response to Ang II in mie y preventing uffering y NO in lood vessels or in the kidney. 3,37 Both effets n inrese the sensitivity of the tuuloglomerulr feedk response, whih my redue the glomerulr filtrtion rte nd use volume retention, whih hs een demonstrted in the development of hypertension in severl experimentl models. 16,38 4 In onlusion, the present study provides evidene tht SOD1 ttenutes renl mirovsulr responses to Ang II y ROS svenging nd mintining NO iovilility. This feture of SOD1 my prevent n erly nd exggerted lood pressure response to Ang II. Furthermore, SOD1 plys n importnt role in regulting renl mirovsulr hypertrophi remodeling. Perspetives Oxidtive stress hs een implited in the pthogenesis of mny humn diseses, inluding hypertension. Emerging evidene suggests tht inresed O 2 levels nd NO defiieny in the kidney ply ruil roles in the development nd persistene of hypertension. SOD1 defiieny leds to endothelil dysfuntion, nd in the present study we demonstrte tht SOD1 defiieny enhnes preglomerulr nd lood pressure responses to Ang II nd is ssoited with renovsulr remodeling nd rdi hypertrophy. From the present study one ould speulte tht inresed O 2 levels in the kidney, euse of ompromised ntioxidnt defense, n use hypertension y inresing preglomerulr vsulr resistne. Studies hve shown tht ptients with endothelil dysfuntion, vsulr remodeling, or rdi hypertrophy re t inresed risk for susequent rdiovsulr events. Therefore, ntioxidnt strtegies speifilly trgeting SOD isoforms ould hve therpeuti enefit in preventing wide spetrum of dverse rdiovsulr outomes. Aknowledgments Crin Nihlén, Krolinsk Institute (Stokholm, Sweden), nd Ulrike Neumnn, Chrité-Universitätsklinikum (Berlin, Germny), re knowledged for tehnil ssistne.

7 Crlström et l SOD1 Limits Remodeling nd Angiotensin II Responses 913 Downloded from y on Otoer 7, 218 Soures of Funding This study ws finnilly supported y the Swedish Reserh Counil (K29-64X nd K29-54X ); the Wllenerg Foundtion; the Swedish Hert nd Lung Foundtion (29264); Wenner-Gren Foundtion; the Swedish Soiety of Mediine; the Mgnus Bergvll Foundtion; the Swedish Soiety of Medil Reserh; the Ntionl Hert, Lung, nd Blood Institute of the Ntionl Institutes of Helth (HL68686); nd the Ntionl Institute of Dietes nd Digestive nd Kidney Diseses (DK nd DK-4987) of the Ntionl Institutes of Helth. None. Dislosures Referenes 1. Bedrd K, Kruse KH. The NOX fmily of ROS-generting NADPH oxidses: physiology nd pthophysiology. Physiol Rev. 27;87: Lssegue B, Clempus RE. Vsulr NAD(P)H oxidses: speifi fetures, expression, nd regultion. Am J Physiol Regul Integr Comp Physiol. 23;285:R277 R Wilox CS. Oxidtive stress nd nitri oxide defiieny in the kidney: ritil link to hypertension? 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