Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II induced cardiac, renal, and vascular injury

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1 & 212 Interntionl Society of Nephrology see commentry on pge 619 Aldosterone deficiency nd minerlocorticoid receptor ntgonism prevent ngiotensin II induced crdic, renl, nd vsculr injury Jmes M. Luther 1,2, Pengcheng Luo 1, Zuofei Wng 1, Smuel E. Cohen 3, Hyung-Suk Kim 4, Agnes B. Fogo 2,5 nd Nncy J. Brown 1 1 Division of Clinicl Phrmcology, Deprtment of Medicine, Vnderilt University Medicl Center, Nshville, Tennessee, USA; 2 Division of Nephrology nd Hypertension, Deprtment of Medicine, Vnderilt University Medicl Center, Nshville, Tennessee, USA; 3 University of Cliforni, Irvine School of Medicine, Irvine, Cliforni, USA; 4 Deprtment of Pthology nd Lortory Medicine, University of North Crolin, Chpel Hill, North Crolin, USA nd 5 Deprtment of Pthology, Vnderilt University Medicl Center, Nshville, Tennessee, USA Angiotensin II cuses crdiovsculr injury in prt y ldosterone-induced minerlocorticoid receptor ctivtion, nd it cn lso ctivte the minerlocorticoid receptor in the sence of ldosterone in vitro. Here we tested whether endogenous ldosterone contriutes to ngiotensin II/ slt-induced crdic, vsculr, nd renl injury y the minerlocorticoid receptor. Aldosterone synthse knockout mice nd wild-type littermtes were treted with ngiotensin II or vehicle plus the minerlocorticoid receptor ntgonist spironolctone or regulr diet while drinking.9% sline. Angiotensin II/slt cused hypertension in oth the knockout nd wild-type mice, n effect significntly lunted in the knockout mice. Either genetic ldosterone deficiency or minerlocorticoid receptor ntgonism reduced crdic hypertrophy, ortic remodeling, nd luminuri, s well s crdic, ortic, nd renl plsminogen ctivtor inhiitor-1 mrna expression during ngiotensin II tretment. Minerlocorticoid receptor ntgonism reduced ngiotensin II/slt-induced glomerulr hypertrophy, ut ldosterone deficiency did not. Comined minerlocorticoid receptor ntgonism nd ldosterone deficiency reduced lood ure nitrogen nd restored nephrin immunorectivity. Angiotensin II/slt lso promoted glomerulr injury through the minerlocorticoid receptor in the sence of ldosterone. Thus, minerlocorticoid ntgonism my hve protective effects in the kidney eyond ldosterone synthse inhiition. Kidney Interntionl (212) 82, ; doi:1.138/ki ; pulished online 23 My 212 KEYWORDS: ldosterone; ngiotensin; crdiovsculr; hypertension; renl injury Correspondence: Jmes M. Luther, Deprtment of Medicine, Division of Clinicl Phrmcology, Vnderilt University Medicl Center, 56 RRB, Nshville, Tennessee , USA. E-mil: Jmes.Luther@Vnderilt.edu Received 6 April 211; revised 16 Ferury 212; ccepted 21 Ferury 212; pulished online 23 My 212 Interruption of ngiotensin II (Ang II) signling is n estlished strtegy to reduce crdiovsculr nd renl events in high-risk popultions. 1,2 Clinicl trils lso demonstrte tht the ddition of minerlocorticoid receptor (MR) ntgonism to ngiotensin-converting enzyme (ACE) inhiition reduces mortlity nd crdic nd renl injury, 3 5 suggesting tht ldosterone contriutes to Ang II medited injury or lterntively tht MR mechnisms my in prt e Ang independent. In this regrd, the glucocorticoids corticosterone or cortisol lso ind to nd ctivte the MR, rising the possiility tht MR ntgonism exerts eneficil effects tht re independent of ldosterone or ngiotensin. The oservtion tht MR ntgonism reduces end-orgn dmge even when endogenous ldosterone concentrtions re suppressed further supports such possiility. 6 8 Phrmcologic ldosterone synthse (AS) inhiition with FAD-286 reduces Ang II induced profirotic gene expression nd crdic nd renl injury in rodent models of hypertension. 9,1 Becuse low levels of ldosterone remin in the circultion during tretment with FAD-286, it is not possile to exclude permissive effect of ldosterone on Ang II signling nd tissue injury. In vitro studies lso suggest tht Ang II cn ctivte the MR in the sence of ldosterone. 11,12 To investigte this in vivo, drenlectomy elimintes circulting ldosterone nonselectively, requiring glucocorticoid replcement tht my not mimic endogenous production. The vilility of As-deficient mice (As / ) results in complete ldosterone deficiency without impired glucocorticoid synthesis nd permits investigtors to dissect out ny contriution of ldosterone to Ang II medited injury. 13,14 We hve previously used this model to determine the contriution of endogenous ldosterone to the cute effects of Ang II on inflmmtory gene expression. 13 This study tests the hypothesis tht endogenous ldosterone contriutes to chronic Ang II induced pro-inflmmtory gene expression nd trget orgn dmge vi the MR. Kidney Interntionl (212) 82,

2 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR RESULTS Bsic metolic mesurements Aldosterone-deficient mice (As / ) nd wild-type () littermtes were treted with Ang II (1 mg/h) or vehicle vi osmotic minipump plus the MR ntgonist spironolctone (; 6 mg/kg/dy) or regulr chow for 8 weeks while drinking.9% sline. Totl ody weight nd len ody mss were similr mong the tretment groups. Serum potssium ws similr in the untreted As / mice compred with (5.4±.6 vs. 5.1±.3, P ¼.7), nd mong the tretment groups (Tle 1). Serum sodium, chloride, icronte, nd corticosterone were lso similr mong the groups. Plsm ldosterone incresed during Ang II tretment in mice nd to n even greter extent during tretment. Aldosterone ws elow the detectle limits in ll As / mice (Po.1 vs. ech group). Urinry sodium excretion incresed during Ang II tretment (Tle 1). This response ws lunted in As / during oth norml chow nd tretment. Urinry potssium excretion ws not different mong groups, except for n increse in the As / -Ang II -treted mice. Chnges in urinry N/K excretion prlleled those of urinry sodium excretion. Blood pressure response to Ang II Systolic lood pressure ws similr t seline (11.9±1.8 nd 97.1±2.3 mm Hg in nd As /, respectively; P ¼.13). Vehicle tretment did not ffect systolic lood pressure (Figure 1). Ang II significntly incresed systolic lood pressure in oth nd As / mice (Po.5). The pressor response to Ang II t post hoc testing ws significntly decresed in As / mice in the presence (Po.5) or sence (Po.5) of. Systolic lood pressure ws similr in -treted As / nd mice (P ¼.2). Crdic ssessment Ang II tretment significntly incresed crdic mss in mice, ut not in As / mice (Figure 2). prevented the effect of Ang II on crdic mss in mice. Results were similr when correcting hert weight y either ody weight (not shown) or tii length. Ang II significntly incresed crdic interstitil firosis in, nd this ws prevented in As / mice (Figure 2). Ang II did not significntly increse interstitil firosis in -treted mice. Crdic perivsculr firosis ws mrkedly incresed y Ang II tretment in mice. The effect of Ang II ws not significnt in -treted mice nd ws lmost entirely rogted in As / mice(figure2cndd). Ang II incresed ortic intim medi nd dventitil thickness in mice (Figure 3 c; P ¼.2 for Ang II effect, P ¼.5 for Ang II effect on dventitil thickening). Either or ldosterone deficiency ttenuted ortic pthologic chnges. There ws no dditive effect of in ldosterone-deficient mice. Kidney injury ssessment Blood ure nitrogen incresed significntly during Ang II tretment (Figure 4). Neither nor ldosterone deficiency prevented the Ang II induced rise in lood ure nitrogen; however, comined ldosterone deficiency nd significntly prevented this increse. Ang II incresed urine lumin excretion in mice, nd this effect ws rogted y tretment (Figure 4) or ldosterone deficiency. Urinry lumin excretion ws reduced during tretment in Ang II treted As / mice, lthough this effect ws not significnt (P ¼.7 for As / -Ang II vs. As / -Ang II group). Ang II incresed verge glomerulr dimeter (P ¼.3); this effect ws prevented y (P ¼.1 for Ang II interction) ut not y ldosterone deficiency (Figure 4c). Ang II tretment produced significnt renl injury, s evidenced y tuulr trophy, perivsculr nd interstitil firosis, proteinceous intrtuulr csts, nd occsionl glomerulosclerosis (Figure 5). There ws no effect of tretment on mesngil hypercellulrity, mesngil expnsion, or infiltrting inflmmtory cells. Ang II tretment reduced nephrin immunorectivity similrly in nd As / mice (from 47.5 to 31.7% in vehicle vs. Ang II tretment); restored nephrin immunorectivity in nd As / mice during Ang II tretment (42.2%; Figure 5). Ang II tretment incresed renl rteril medi re (P ¼.4); this ws lso Tle 1 Physiologic mesurements t the end of study protocol -Vehicle Ang II -Vehicle Ang II As / -Vehicle As / Ang II As / -Vehicle As / Ang II Body weight (g) 28.± ± ± ± ± ± ± ±1. Serum N (meq/l) 147.7± ± ± ± ± ± ± ±1.1 Serum K (meq/l) 5.7± ± ± ±.5 5.4± ± ± ±.29 HCO 3 (meq/l) 2.1± ± ± ± ± ± ± ±1.33 Aldosterone (pg/ml) 373.± ± ± ±458 ND # ND # ND # ND # Corticosterone (ng/ml) 33.7± ± ± ± ± ± ± ±62.7 Urine N V(mmol/dy) 143± ± ± ± ±126 15±428 w 1286± ±148 z Urine K V(mmol/dy) 649±1 917±56 633± ±13 71±63 776±11 781± ±426,y Urine N/K rtio 1.6±.1 4.7± ±.4 4.7±2.5 1.±.1 2.2±.8 1.6±.2 1.6±.2 w Urine Al/Cr (mg/g) 25.9± ± ± ±12.1 w 13.± ±114.7 w 32.5± ±6. w Arevitions: Al/Cr, lumin/cretinine; Ang II, ngiotensin II; As /, ldosterone synthse deficient; ND, elow detectle limit;, spironolctone;, wild type. Results re men±s.e.m. Po.5 vs. -vehicle, w Po.5 vs. Ang II, y Po.5 vs. As / -vehicle, z Po.5 vs. Ang II, # Po.5 vs. ll tretment groups. 644 Kidney Interntionl (212) 82,

3 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR SBP (mm Hg) SBP (mm Hg) vehicle vehicle Time (weeks) Time (weeks) Regulr chow Spironolctone Figure 1 Effect of tretment on lood pressure. Systolic lood pressure (SBP) during regulr chow () nd spironolctone chow (). Angiotensin II (Ang II) incresed SBP significntly in wild-type (; J) nd ldosterone synthse deficient (As / ;.) mice, ut the lood pressure response ws ttenuted in As /.Po.5 vs. -vehicle; w Po.5 vs. Ang II. Hert weight (mg/mm tii length) d Crdic interstitil firosis (re %) c Crdic perivsculr firosis score Vehicle Vehicle Spironolctone Figure 2 Aldosterone deficiency nd MR ntgonism ttenute crdic hypertrophy nd perivsculr firosis. Angiotensin II (Ang II) incresed () crdic mss nd cused () crdic interstitil firosis nd (c) perivsculr firosis in wild-type () mice. Crdic hypertrophy ws similrly rogted in spironolctone ()-treted nd ldosterone synthse deficient (As / ) mice. Interstitil nd perivsculr firosis were ttenuted nd not significntly incresed in -treted mice nd were prevented in As / mice. (d) Representtive imges of crdic perivsculr firosis re shown for ech tretment group; Msson trichrome (originl mgnifiction 4), Br ¼ 2 mm. Po.5 vs. -vehicle; w Po.5 vs. Ang II. MR, minerlocorticoid receptor. ttenuted y (P ¼.2), ut not ldosterone deficiency (Supplementry Figure S1 online). The increse in dventitil re in response to Ang II ws prevented y tretment (P ¼.3), ut ws not ffected y ldosterone deficiency (Supplementry Figure S1 online). In contrst, there ws no effect of either or ldosterone deficiency on Ang II induced renl firosis or glol kidney injury score (Supplementry Figure S1c nd d online). Profirotic gene expression Ang II significntly incresed mrna expression of plsminogen ctivtor inhiitor-1 (Pi-1) within crdic, renl, nd ortic tissues (Figure 6). Either or ldosterone deficiency prevented the increse of Pi-1 mrna expression in these tissues to vrying degrees. Crdic preproendothelin-1 (Et-1) expression ws unchnged (Supplementry Figure S2 c online). Ang II incresed Et-1 expression within renl tissue, Kidney Interntionl (212) 82,

4 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR Aort intim medi thickness (µm) c Aort dventiti thickness (µm) Vehicle Vehicle Spironolctone Figure 3 Aldosterone deficiency nd MR ntgonism ttenute ortic hypertrophy nd perivsculr firosis. Angiotensin II (Ang II) incresed ortic () intim medi thickness nd () dventitil thickness in vehicle-treted wild-type () mice, ut not in spironolctone ()-treted or ldosterone synthse deficient (As / ) mice. (c) Representtive imges re provided; Msson trichrome stin (originl mgnifiction 4), Br ¼ 1 mm. Po.5 vs. -vehicle, w Po.5 vs. Ang II. c 4 BUN (mg/dl) Urine microlumin (mg Al per g Cret) Figure 4 Endogenous ldosterone contriutes to ngiotensin II-induced glomerulr injury. Angiotensin II (Ang II) cused n increse in () lood ure nitrogen (BUN), () luminuri, nd (c) glomerulr enlrgement in wild-type () mice, nd BUN elevtion nd glomerulr enlrgement in ldosterone synthse deficient (As / ) mice. Only comined spironolctone nd genetic ldosterone deficiency reduced Ang II induced BUN elevtion. Spironolctone () prevented luminuri in Ang II treted mice. As / mice were lso protected ginst Ang II induced luminuri. prevented glomerulr expnsion, wheres ldosterone synthse deficiency did not. Po.5 vs. -vehicle; w Po.1 vs. Ang II; z Po.5 vs. As / -vehicle; 8 Po.5 vs. -Ang II-; y Po.5 vs. As / -Ang II. Avg. glomerulr dimeter (µm) nd this effect ws prevented y ut not y ldosterone deficiency. In contrst, ldosterone deficiency ws ssocited with incresed Et-1 expression in the ort. Renl 11hsd gene expression We nlyzed renl mrna expression of 11-hydroxysteroid dehydrogense (11hsd)-1 nd 11hsd-2 gene expression during Ang II dministrtion. We oserved no significnt effect of Ang II on either 11hsd-1 (1.12±.2-fold chnge vs. vehicle control; P ¼.84) or 11hsd-2 (1.18±.2; P ¼ 1., n ¼ 5 for ech group). DISCUSSION We found tht endogenous ldosterone contriutes to Ang II/slt induced crdic hypertrophy, crdic firosis, nd ortic remodeling through n MR-dependent mechnism. In contrst, in the kidney, Ang II/slt cn lso induce glomerulr injury nd rteriolr hypertrophy vi ctivtion of the MR, even in the sence of endogenous ldosterone. Ang II/slt cuses renl interstitil firosis through n MR- nd ldosterone-independent mechnism. Numerous studies hve demonstrted tht ldosterone dministrtion cuses crdic nd ortic inflmmtion nd 646 Kidney Interntionl (212) 82,

5 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR Vehicle Vehicle Spironolctone Figure 5 Renl histology nd nephrin immunorectivity. Representtive imges of kidney histologic injury re shown for ech tretment group (); periodic cid Schiff (PAS) stin. Angiotensin II (Ang II) tretment decresed () nephrin immunorectivity in wild-type () nd ldosterone synthse deficient (As / ) mice. Originl mgnifiction 4, Brs ¼ 1 mm. remodeling, wheres MR ntgonism prevents inflmmtion nd firosis in slt-treted rodents Becuse endogenous ldosterone concentrtions re suppressed during high-slt intke, nd lignds other thn ldosterone cn ctivte the MR, the solute requirement for endogenous ldosterone in MR-medited pthology hs een clled into question. Compelling studies in rts during excessive sodium intke suggest n ldosterone-independent, MR-dependent mechnism of crdiovsculr nd renl injury. For exmple, MR ntgonism reduces crdic hypertrophy in the Dhl slt-sensitive rt, low-ldosterone model of hypertension, nd the uthors speculted tht ctivtion of the MR y glucocorticoids contriutes to this effect. 6 In the hert, glucocorticoids my normlly function s MR ntgonists under norml conditions, ut contriute to MR ctivtion depending on the redox stte of the cell. 21 The MR lso contriutes to distolic dysfunction nd oxidtive stress during low-dose Ang II dministrtion, wheres ldosterone concentrtions were unchnged. 22 In contrst, we oserved tht either MR ntgonism or genetic ldosterone deficiency ttenuted crdic hypertrophy nd crdic perivsculr injury, demonstrting pivotl role for endogenous ldosterone in this model. These dt re consistent with other studies testing the effect of phrmcologic ldosterone synthse inhiition, nd suggest tht endogenous ldosterone contriutes sustntilly to Ang II induced crdic injury. 9,1,23 We hve reported previously tht or phrmcologic ldosterone synthse inhiition with FAD-286 meliorted Ang II/slt induced crdic hypertrophy nd interstitil firosis ut not coronry perivsculr firosis in rt model. 1 In this study, genetic ldosterone deficiency or prevented perivsculr firosis s well s interstitil firosis nd hypertrophy, suggesting tht the lck of effect in the prior study my hve een ecuse of residul ldosterone production during phrmcologic inhiition. The modest nd vrile effect of on interstitil nd perivsculr firosis in the hert my lso reflect the use of low-dose. The eneficil effect of high doses of on these mesures is well estlished. 24 We hve lso demonstrted previously tht either FAD- 286 or prevents Ang II induced lood ure nitrogen elevtion, glomerulosclerosis, tuulointerstitil firosis, nd perivsculr remodeling in rt model. In this study, lthough Ang II cused significnt renl injury, glomerulosclerosis ws rre, nd overll renl injury ws not severe, likely reflecting species nd strin difference. Nevertheless, MR ntgonism hd selective eneficil effects on glomerulr injury, eyond the effect of ldosterone deficiency, suggesting tht ctivtion of the MR y nonldosterone lignds cn contriute to injury. Tht MR ntgonism decreses Ang II induced renl injury hs een estlished previously. In humns with dietic nephropthy, for exmple, MR ntgonism reduces Kidney Interntionl (212) 82,

6 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR Pi-1 reltive mrna expression Pi-1 reltive mrna expression Hert Pi-1 reltive mrna expression 6 c 4 2 Kidney proteinuri during mximl ACE inhiition nd my e more effective thn dditionl renin ngiotensin ldosterone system lockde with n Ang II receptor ntgonist. 3 In rts treted with Ang II nd nitric oxide synthse inhiitor, drenlectomy or MR ntgonism with eplerenone prevents proteinuri nd renl rteriolr nd glomerulr injury. 25 MR ntgonism lso protects ginst glomerulr injury in sltsensitive rts in which plsm ldosterone is suppressed, primrily vi preserved podocyte function. 7,8,26 Aort Figure 6 Tissue plsminogen ctivtor inhiitor-1 mrna expression. Angiotensin II (Ang II) tretment incresed plsminogen ctivtor inhiitor-1 (Pi-1) mrna levels in () crdic, () ortic, nd (c) renl tissues in wild-type () mice. Spironolctone () or genetic ldosterone synthse deficiency (As / ) prevented this increse within the hert nd ort, nd ttenuted the effect within the kidney. Po.5 vs. -vehicle; w Po.5 vs. -Ang II; z Po.5 vs. As / -vehicle-. To our knowledge, however, no studies hve demonstrted the pthologic effects of Ang II in vivo vi MR ctivtion, while excluding the possiility of endogenous ldosterone synthesis. Fctors other thn ldosterone cn ctivte the MR in mesngil cells nd podocytes, contriuting to proteinuri nd mesngil expnsion. For instnce, Rc1 GTPse contriutes to podocyte injury vi ldosterone-independent MR ctivtion, producing proteinuri nd renl injury. 27 Conditions such s hyperglycemi, oesity, nd slt loding my ctivte the Rc1-MR pthwy nd contriute to injury. 27,28 The clssicl MR or novel memrne-ound MR my e responsile for this ldosterone-independent signling. 29 Our dt suggest tht other fctors lso ct vi the MR to produce glomerulr expnsion. Glucocorticoids cn ind to nd ctivte the MR. Normlly, glucocorticoids re inctivted y 11HSD-2 in ldosterone trget cells. 21 However, Rfiq et l. 3 recently reported tht hydrocortisone cuses renl injury in drenlectomized rts through n MR-dependent mechnism, which could result from prtil gonism of the MR in the sence of endogenous ldosterone or from stimultion of the MR on nonepithelil cells. There ws no significnt difference in corticosterone concentrtions to explin our findings. Alterntively, metolic products of 11HSD-2 could ct s physiologic MR ntgonists or lter the redox stte of the cell. 21,31 Glomerulr 11hsd-2 mrna expression hs een descried in mesngil cells nd podocytes. 32,33 We found no effect of Ang II on either 11hsd-1 or 11hsd-2 gene expression in the kidney, lthough we did not exclude n effect on 11HSD-2 ctivity. Other investigtors hve lso demonstrted tht Ang II ctivtes the MR vi rective oxygen species or NF-kB, providing nother possile explntion for these findings. 22,34 In contrst to the protective effect of MR ntgonism on Ang II induced glomerulr expnsion nd nephrin immunorectivity, neither MR ntgonism with nor ldosterone deficiency prevented Ang II/slt induced renl interstitil firosis in this study. These findings re consistent with n AT 1 receptor dependent profirotic effect of Ang II, s other investigtors hve demonstrted. 35 The dt re lso consistent with the finding tht decreses glomerulr injury, ut not interstitil firosis. 8 Multiple studies hve demonstrted the centrl importnce of PAI-1 s profirotic meditor in renl, vsculr, nd crdic tissues. 36 Although Ang II is clssic stimulus for Pi-1 gene expression, ldosterone nd the MR re essentil for mximl response in certin tissues. We reported previously tht endogenous ldosterone contriutes to the effect of cute Ang II infusion on Pi-1 mrna expression within the hert, ut not in ort. 13 During chronic Ang II exposure, endogenous ldosterone ppers to contriute to incresed Pi-1 expression within oth the hert nd the ort vi the MR. The concordnt pthologic effects suggest centrl role for ldosterone, the MR, nd PAI-1, nd suggest tht improvement in our model could e ccomplished vi reduction of Pi-1 expression. 648 Kidney Interntionl (212) 82,

7 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR Finlly, genetic ldosterone deficiency resulted in lunted pressor response to Ang II; decresed lood pressure my hve contriuted to the protective effect of ldosterone deficiency on crdiovsculr remodeling. This underscores the lck of protective effect of ldosterone deficiency on glomerulr expnsion. Similrly, the pressor response to Ang II ws similr in As / -Ang II nd - Ang II groups, suggesting tht differences etween the effects of MR ntgonism nd ldosterone deficiency on renl injury were lood pressure independent. Renin ngiotensin ldosterone system lockde with either ACE inhiitors or Ang II receptor ntgonists prevents crdiovsculr nd renl events in high-risk individuls, nd provides dditionl enefit eyond ACE inhiition. 3 5 Although ACE inhiition reduces circulting Ang II levels, other enzymes such s chymse contriute to locl Ang II genertion vi n lterntive pthwy. 37 Although it is well estlished tht ldosterone produces MR-medited injury, other Ang II induced lignds my lso ctivte the MR nd contriute to tissue injury. This study demonstrtes tht ldosterone medites portion of Ang II induced crdic, renl, nd vsculr injury nd profirotic gene expression. The dditionl enefit of MR ntgonism in the setting of ldosterone deficiency suggests tht Ang II cn lso medite MR-dependent effects in vivo in the sence of ldosterone. Therefore, MR ntgonism my provide dditionl eneficil effects regrdless of circulting ldosterone concentrtions. MATERIALS AND METHODS Animls All experiments were reviewed nd pproved y the Vnderilt University Institutionl Animl Cre nd Use Committee. As / mice were generted on 129 ckground 38 nd ckcrossed over seven genertions onto the C57Bl6/J strin (Jckson Lortory, Br Hror, ME). Mice were genotyped y rel-time PCR (Applied Biosystems 79HT, Foster City, CA) using Tqmn proes for sequence in the Cyp112 gene nd for portion of the gene contined in the neomycin-resistnce cssette s previously descried. 13 Mle As / nd littermtes were studied. Animls were housed in temperture-controlled fcility with 12-h light/ drk cycle. Mice were mintined on.9% sline in the drinking wter d liitum strting t ge 8 weeks nd continued for the durtion of the study. Chronic Ang II infusion protocol At 12 weeks of ge, mice were rndomized to tretment with Ang II,, or comintion of oth. Ang II (1 mg/h, ClBiochem, L Joll, CA) or vehicle were delivered y osmotic minipumps (Alzet Model 21, Alz, Plo Alto, CA) implnted under pentoritl nesthesi (5 mg/kg intrperitonelly) t 12 weeks of ge. This dose of Ang II ws chosen to induce modest hypertension nd injury in mice. 39 After pump implnttion, mice received either stndrd mouse chow (Purin Lortory Rodent 51, St Louis, MO) or stndrd chow supplemented with, continued for the durtion of the study. chow ws formulted y TestDiet (Richmond, IN) t concentrtions to provide 6 mg/kg/dy sed on prior studies demonstrting tht this dose decreses end-orgn dmge. 1,4 Minipumps were replced fter 4 weeks. Mice were killed 8 weeks fter initil minipump implnttion (2 weeks of ge). Cervicl disloction ws performed, the left renl rtery ws clmped, nd lood ws collected y crdic puncture nd trnsferred into tue contining dipotssium-edta tues (Microvette CB K2E, Srstedt AG, Numrecht, Germny). The se of the hert, the first 2 mm of descending ort, nd coronl sections of the kidney were fixed in 4% uffered prformldehyde overnight, trnsferred to 7% ethnol, nd then emedded in prffin for histologicl evlution. The reminder of the hert, liver, nd kidney were snp-frozen in liquid nitrogen for mrna extrction. The remining ort (descending ort to ove the renl rteries) ws trnsferred into fresh phosphte-uffered sline solution, stripped of dventitil tissue, collected into RNALter solution (Amion, Austin, TX) t 4 1C overnight, nd then trnsferred to vil for storge t 8 1C. Crdiovsculr mesurements Blood pressure ws mesured every 2 weeks strting t 6 8 weeks of ge (X4 weeks efore rndomiztion) using utomted til-cuff impednce plethysmogrphy (BP-2 Blood Pressure Anlysis System; Visitech Systems, Apex, NC) in unnesthetized, trined mice, pre-wrmed for 5 min t 37 1C. At 2 weeks of ge, trnsthorcic echocrdiogrphy ws performed in conscious mice using 15-MHz trnsducer (Sonos 55 system, Agilent, Andover, MA) s previously pulished. 41 Blood nd urine chemistry Blood ws otined for chemistry fter 8 weeks of tretment, efore killing. Mice were fsted in clen cge t 9 h, nd 5 ml whole lood ws collected t h vi sphenous vein into heprinized cpillry tue (Microvette CB K2E, Srstedt AG) nd processed immeditely y istat EC8 þ crtridge (Hesk, Lovelnd, CO). An dditionl 3 ml of lood ws collected into dipotssium- EDTA (Microvette CB K2E, Srstedt AG) nd centrifuged t 6 r.p.m. for 5 min. Plsm ws stored t 8 1C until ssy. Aldosterone ws determined using rdioimmunossy utilizing 125 I-ldosterone (MP Biomedicls, Irvine CA), primry ntiody to ldosterone (NIDDK Ntionl Hormone & Peptide Progrm, Torrnce, CA), nd secondry nti-rit g-gloulin ntiody (Linco Reserch, St Chrles, MO). Corticosterone ws mesured using commercilly ville rdioimmunossy kit (ImmuChem Doule Antiody Corticosterone Kit, MP Biomedicls). For urine collection, mice were housed in individul metolic cges (Nlgene Lwre, Rochester, NY) for 24 h. Urine ws collected while mice were ingesting norml chow nd.9% sline d liitum during the finl week of tretment (tht is, Ang II, ). Urine N þ nd Cl were mesured y flme photometry (ILS 94). Urine ws nlyzed for lumin using n immunoturidimetric ssy nd for cretinine y modified colorimetric ssy tht correltes well with HPLC-sed methods (DCA 2 þ Anlyzer nd Microlumin/Cretinine crtridges, Byer, Elkhrt, IN). 42 Histopthologic nlysis Imges were cptured on Zeiss AxioScop 4 using MRGr 1. (Medi Cyernetics, Silver Spring, MD). All quntittive imge nlyses were performed using ImgeJ, version 1.4g (NIH, Bethesd, MD), 43 nd ll imge mesurements were clirted using 2 mm slide micrometer photogrphed under pproprite mgnifiction. Glomerulr re nd dimeter were determined y outlining 3 5 consecutive glomeruli per niml (imged using 4 Kidney Interntionl (212) 82,

8 JM Luther et l.: Aldosterone-independent Ang II injury vi the MR ojective), for 3 4 nimls per group. Renl medi nd perivsculr re were mesured y trcing the inner lumen, outer order of the tunic medi, nd outer order of the dventitil re ( 5 ojective, 3 5 interloulr rteries per niml). Collgen re ws quntified y picro-sirius red stining imged under cross-polrized light, mesured s percentge of collgen-positive re, excluding hilr vessels from the nlysis. Aortic intim medi nd dventiti thickness re verges from X4 liner mesurements per ort. An investigtor (ABF) linded to tretment ssignment lso ssessed histopthology. Kidney injury ws scored semiquntittively on Msson trichrome stined sections, from to 3 for ech high-power field ( indicting no injury; 1 indicting single smll focus of firosis; 2 indicting severl smll foci; 3 indicting multifocl firosis). Crdic interstitil firosis ws scored semiquntittively s n estimte of percentge of firotic re. Crdic perivsculr firosis ws scored semiquntittively ( indicting no firosis; 1 indicting dventitil firosis less thn medi re; 2 indicting firosis equl to medi re; nd 3 indicting firosis greter thn medi re). Nephrin immunohistochemistry ws performed fter citrte uffer ntigen retrievl, using n nti-mouse nephrin primry ntiody (R&D Systems AF3159, Minnepolis, MN), iotinylted secondry ntiody (Vector Ls BA-5, Burlingme, CA), nd detection with the VECTASTAIN ABC kit (Vector Ls). Nephrinpositive glomerulr re ws mesured in 15 2 glomeruli per niml using ImgeJ softwre. Gene expression Crdic, heptic, nd renl totl RNAs were extrcted using RNA Wiz (Amion) nd RNesy Midi Kit (Qigen, Vlenci, CA), nd ortic RNA ws extrcted with RNesy Mini Kit (Qigen). Reverse trnscription ws performed using TqMn Reverse Trnscription Kit (Applied Biosystems, Brnchurg, NJ). Quntittive rel-time PCR ws performed in duplicte on the icycler iq Multi-Color Rel Time PCR Detection System (Bio-Rd, Hercules, CA) using iq SYBR Green Supermix (Bio-Rd) using primers s previously descried nd 5 ml totl rection volume. 39 Templte cdna used in the rection ws 2 mg for hert nd kidney nd 5 ng for ort. Experimentl cycle threshold (C t ) vlues were normlized to -ctin mesured on the sme plte, nd fold differences in gene expression were determined using the 2 DDCt method. 44 Sttistics Dt re presented s men±s.e.m. Comprisons mong tretment groups were performed using nlysis of vrince, with lest significnt difference post hoc testing for etween-group pirs. Becuse not ll pir-wise comprisons re of physiologic relevnce nd normlity could not e ssumed, we confirmed significnt etween-group differences using nonprmetric tests. Wilcoxon signed-rnk test nd Wilcoxon rnk-sum test were used for pired nd unpired comprisons, respectively, for ny nonnormlly distriuted dt. Individul drug effect nd drug genotype interction for injury mesures were ssessed y multivrile liner regression with genotype, Ang II tretment, nd tretment s exposure vriles. All sttisticl nlyses were performed using SPSS for Windows, version 17. (SPSS, Chicgo, IL), or the open source sttisticl pckge R, version A two-tiled P-vlue of o.5 ws considered significnt. DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS This work ws supported y the NIH (DK81662, HL696, DK79341, DK56942, nd DK44757), nd the Vnderilt Mouse Metolic Phenotyping Center (DK59637). Steroid hormone ssys were performed y the Vnderilt Dietes Reserch nd Trining Center Hormone Assy Core L (DK2593). SUPPLEMENTARY MATERIAL Figure 1. Interloulr rtery medi (A) nd dventitil re (B) incresed during ngiotensin (Ang) II (P ¼.4), nd this ws ttenuted y spironolctone () (P ¼.2), ut not ldosterone deficiency. Figure 2. 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