1521-0103/358/3/548–557$25.00 http://dx.doi.org/10.1124/jpet.116.234765THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:548–557, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

CS-3150, a Novel Nonsteroidal Mineralocorticoid ReceptorAntagonist, Shows Preventive and Therapeutic Effects On RenalInjury in Deoxycorticosterone Acetate/Salt-InducedHypertensive Ratss

Kiyoshi Arai, Yuka Morikawa, Naoko Ubukata, Hiroyuki Tsuruoka, and Tsuyoshi HommaEnd-Organ Disease Laboratories (K.A., N.U., T.H.), Rare Disease and LCM Laboratories (Y.M.), and Venture Science Laboratories(H.T.), Daiichi Sankyo Co., Ltd., Tokyo, Japan

Received May 5, 2016; accepted June 30, 2016

ABSTRACTThe present study was designed to assess both preventiveand therapeutic effects of (S)-1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidal mineralocorticoidreceptor antagonist, on renal injury in deoxycorticosterone acetate(DOCA)/salt-induced hypertensive rats (DOCA rats). From 7weeksof age, DOCA was subcutaneously administered once a weekfor 4 weeks to uninephrectomized rats fed a high-salt diet. Inexperiment 1, CS-3150 (0.3–3 mg/kg) was orally administeredonce a day for 4 weeks coincident with DOCA administration. Inexperiment 2, after establishment of renal injury by 4 weeks ofDOCA/salt loading, CS-3150 (3 mg/kg) was orally administeredonce a day for 4 weeks with or without continuous DOCAadministration. In experiment 1, DOCA/salt loading significantlyincreased systolic blood pressure (SBP), which was prevented

by CS-3150 in a dose-dependent manner. Development ofrenal injury (proteinuria, renal hypertrophy, and histopathologicalchanges in glomeruli and tubule) was also suppressed byCS-3150with inhibition of mRNA expression of fibrosis, inflammation, andoxidative stress markers. In experiment 2, under continuousDOCA treatment, CS-3150 clearly ameliorated existing renal injurywithout lowering SBP, indicating that CS-3150 regressedrenal injury independent of its antihypertensive action. Moreover,CS-3150 treatment in combination with withdrawal of DOCAshowed further therapeutic effect on renal injury accompaniedby reduction in SBP. These results demonstrate that CS-3150not only prevents but also ameliorates hypertension and renalinjury in DOCA rats. Therefore, CS-3150 could be a promisingagent for the treatment of hypertension and renal disorders, andmay have potential to promote regression of renal injury.

IntroductionMineralocorticoid receptor (MR) was originally recognized

to be expressed in epithelial cells of the distal tubule andcollecting duct in the kidney, and to play a role in theregulation of electrolyte homeostasis, body fluid, and bloodpressure (Funder, 1995). In addition to such classic actions, ithas been reported that MR is also expressed in other cells inthe kidney, such as podocytes (Shibata et al., 2007), mesangialcells (Nishiyama et al., 2005), and fibroblasts (Nagai et al.,2005), and MR activation by endogenous ligands (e.g., aldo-sterone) is involved in the pathogenesis of renal disorder viadirect enhancement of fibrosis (Brem et al., 2011), inflamma-tion (Siragy and Xue, 2008), and oxidative stress (Patni et al.,2007). In fact, numerous clinical studies using spironolactoneand eplerenone, which are clinically available MR antago-nists, have demonstrated that MR blockade is an effective

strategy for treatment of chronic kidney disease (Sato et al.,2005; Mehdi et al., 2009; Boesby et al., 2011) as well ashypertension (Chapman et al., 2007; Calhoun andWhite, 2008).Recently, we discovered (S)-1-(2-hydroxyethyl)-4-methyl-N-

[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidalMR antagonist. CS-3150 is a selective and highly potent MRantagonist with long-lasting MR antagonism after oral ad-ministration (Arai et al., 2015). It is well known thatdeoxycorticosterone acetate (DOCA), a synthetic mineralocor-ticoid, induces hypertension and renal injury in combinationwith salt loading to rats (DOCA rats), and this hypertensiverat is widely used as an MR-mediated disease model toevaluate pharmacological effects of MR antagonists or otherdrugs (Klanke et al., 2008; Seifi et al., 2010). We havepreviously reported that CS-3150 inhibits elevation in systolicblood pressure (SBP) in DOCA rats (Arai et al., 2015);however, we have not examined the effects of CS-3150 onrenal injury. In addition, it remains unclear whether CS-3150elicits therapeutic efficacy in the established hypertension

dx.doi.org/10.1124/jpet.116.234765.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: CMC, carboxymethylcellulose; Col1a1, collagen 1a1; CS-3150, (S)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide; DOCA, deoxycorticosterone acetate; IL-6, interleukin-6; MC, methylcellulose; MR,mineralocorticoid receptor; PAI-1, plasminogen activator inhibitor-1; SBP, systolic blood pressure; UP, urinary protein; UV, urinary volume;WKY/Izm, Wistar Kyoto.

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and renal injury in DOCA rats. Therefore, in the presentstudy, two independent experiments were conducted to in-vestigate the preventive and therapeutic effects of CS-3150 onhypertension and renal injury in DOCA rats.

Materials and MethodsTest Compounds. CS-3150 was synthesized at Medicinal Chem-

istry Research Laboratories, Daiichi Sankyo Co., Ltd. (Tokyo, Japan),as described in patent no. WO2008126831. The chemical structure ofCS-3150 is shown in Fig. 1.

Experimental Protocol. All procedures involving animal use inthese experiments were carried out in accordance with the guidelinesof the Institutional Animal Care and Use Committee of Daiichi SankyoCo., Ltd. Five-week-old male Wistar Kyoto (WKY/Izm) rats werepurchased from Funabashi Farm Co., Ltd. (Chiba, Japan). The ratsweremaintained in a room at 506 10% relative humidity and 236 2°Cunder a 12-hour light/dark cycle, and were allowed free access to food(FR-2; Funabashi Farm Co., Ltd.) and water.

In experiment 1 (preventive study), 6-week-old maleWKY/Izm ratswere anesthetized with isoflurane inhalation, and the left kidney wasremoved. After 1-week recovery (at 7 weeks of age), the rats weredivided into five groups: control, vehicle, and three groups of CS-3150treatments. A 20-mg/kg dose of DOCA, suspended in 0.5% carboxy-methylcellulose (CMC; Wako Pure Chemical Industries, Ltd., Osaka,Japan), was subcutaneously administered once a week for 4 weeks. Incontrol rats, 0.5% CMC was administered instead of DOCA. The ratswere fed a high-salt diet (FR-2 containing 4% NaCl; Funabashi FarmCo., Ltd.) during the study. To assess the preventive effect of CS-3150,vehicle (0.5% methylcellulose [MC]; Wako Pure Chemical Industries,Ltd.) or CS-3150 (0.3, 1, and 3 mg/kg) was orally administered once aday for 4 weeks.

In experiment 2 (therapeutic study), hypertension and renal damagewere established by 4-week DOCA/salt loading as conducted inexperiment 1. At 11 weeks of age, the control rats (0.5% CMC–treatedrats) were divided into two subgroups, and the DOCA-treated rats weredivided into five subgroups. One of each subgroup was dissectedaccording to the following procedure as the baseline. Two subgroups ofDOCA-treated rats received a further 4-week administration of DOCA,whereas the other two received 0.5% CMC instead. To assess thetherapeutic effect of CS-3150, vehicle (0.5% MC) or CS-3150 (3 mg/kg)was orally administered once a day for 4weeks. Regarding a subgroup ofcontrol rats, 0.5%CMCwas received once aweek, and vehicle (0.5%MC)was orally administered once a day for a further 4 weeks. All rats werefed a high-salt diet (FR-2 containing 4% NaCl; Funabashi Farm Co.,Ltd.) during the study.

Measurement of Blood Pressure and Urinary Parameters.SBP was measured by the tail-cuff method (BP-98A; Softron Co., Ltd.,Tokyo, Japan) in awake, restrained animals at 7, 9, and 11 weeks of agein experiment 1, and at 7, 11, 13, and 15 weeks of age in experiment 2.

Rats were individually placed in a metabolic cage, and urine wascollected for 24 hours at 7 and 11 weeks of age in experiment 1, and at7, 11, 13, and 15 weeks of age in experiment 2. Urinary volume (UV)was measured, and the urine samples were centrifuged (1820 � g for15 minutes). The supernatant was used for measurement of protein

concentration by an automated clinical analyzer (BiOLiS 24i Pre-mium; TokyoBoekiMachinery Ltd., Tokyo, Japan), and urinary protein(UP) excretion for 24 hours was calculated. Urinary sodium (Na1) andpotassium (K1) concentrations were also measured using an electrolyteanalyzer (STAX-2; Techno Medica Co., Ltd., Kanagawa, Japan), andboth excretions for 24 hours were calculated (Supplemental Figs. 1 and 2).In addition, urinary MCP-1 concentration was measured by an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN), andits excretion for 24 hours was calculated (Supplemental Tables 1 and 2).

Necropsy. In experiment 1, at 11 weeks of age, each rat wasanesthetized by isoflurane inhalation, and after blood sampling fromthe abdominal aorta, the right kidneywas excised andweighed. A partof the right kidney was placed in RNA stabilization reagent (RNA-later; Invitrogen, Carlsbad, CA) and stored at 280°C until total RNAisolation. The rest of the right kidney was sectioned into 2- to 3-mmslices and fixed in 10% neutral buffered formaldehyde solution (WakoPure Chemical Industries, Ltd.). In experiment 2, necropsywas performedat 11weeks of age in someof the rats as the baseline andat 15weeks of ageon the remaining rats, as described earlier.

Relative mRNA Expression Analysis by Real-Time Poly-merase Chain Reaction. Relative gene expression in the kidneywas determined by quantitative real-time polymerase chain reaction(PCR) as follows. Total RNA was extracted using TRIzol Reagent(Invitrogen), and the cDNA was synthesized by a First-Strand cDNASynthesis Kit (GE Healthcare, Buckinghamshire, UK) with randomhexamer primers. Quantitative PCR was performed using the cDNAbyTaqManGeneExpressionAssays (AppliedBiosystems, Foster City,CA) and TaqMan universal PCR master mix according to the manu-facturer’s instructions. The following assays were used: TGF-b1(Rn00572010_m1), collagen 1a1 (Col1a1; Rn01463848_m1), interleukin-6(IL-6; Rn01410330_m1),MCP-1 (Rn00580555_m1), p47phox (Rn00586945_m1),p67phox (Rn01759079_m1), and peptidylprolyl isomerase B(Rn03302274_m1). PCR reactions were performed using a 7900 HT FastReal Time PCR system (Applied Biosystems). The data were analyzedautomatically using SDS 2.4 software (Applied Biosystems) to determinethe expression levels. Relative mRNA levels of each gene were calculatedby normalizing them to the peptidylprolyl isomerase B mRNA level.

Histopathological Examination of Kidney. The excised rightkidney fixed in 10% neutral buffered-formaldehyde solution wasembedded in paraffin. Sections were stained with periodic acidmethenamine silver for the following histopathological examination.Severity of glomerulosclerosis was semiquantitatively evaluated in30 glomeruli in each specimen according to the criteria developed byUehara et al. (1993). The extent of glomerulosclerosis was graded from0 to 4 as follows: 0, normal; 1,,25% of sclerosis; 2, 25–50% of sclerosis;3, 50–75% of sclerosis; and 4, .75% of sclerosis. Severity of tubularinjury of both the cortex and medulla was also semiquantitativelyevaluated in 10 randomly selected microscope fields (�200) in eachspecimen according to the criteria developed by Uehara et al. (1993).The extent of tubular injury was graded from 0 to 4 as follows: 0, nolesion; 1, very mild focal dilatation of tubules; 2, large number ofdilated tubules with widening of interstitium; 3, fairly extensivedilatation of tubules with cystic formation and widening of intersti-tium; and 4, complete atrophy of tubules.

Statistical Analysis. Data are expressed as the mean 6 S.E.M.The comparison between two groups was evaluated by Student’s t testor Aspin-Welch’s t test. For multiple comparisons, Dunnett’s test wasperformed. A P value of less than 0.05 was considered to be statisticallysignificant. All calculations were conducted using SAS System Release8.2 and 9.2 (SAS Institute Inc., Tokyo, Japan).

ResultsExperiment 1

SBP and UP Excretion. DOCA/salt loading to WKY/Izmrats induced progressive elevation of SBP (Fig. 2A). Treatmentwith CS-3150 suppressed SBP elevation in a dose-dependentFig. 1. Chemical structure of CS-3150.

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manner, and the antihypertensive effect at 11 weeks of age wassignificant at 1 and 3 mg/kg (Fig. 2A). UP excretion was alsoincreased in the vehicle group, and was prevented by CS-3150(Fig. 2B). Interestingly, 1 mg/kg CS-3150 completely blockedthe increase in UP excretion while only partially suppressingSBP elevation (Fig. 2, A and B).Other Urinary Parameters. UV was increased in the

vehicle group, and was prevented by CS-3150 (SupplementalFig. 1A). Urinary Na1 and K1 excretions were also increasedin the vehicle group; however, there were no significantdifferences between the vehicle- and CS-3150–treated groups(Supplemental Fig. 1, B and C).Urinary MCP-1 excretion was increased in the vehicle, and

was prevented by CS-3150 (Supplemental Table 1).Kidney Weight and Histology. Kidney weight was

markedly increased in the vehicle group (Fig. 3A). In addition,DOCA/salt loading to rats resulted in marked glomerular andtubular injury, such as glomerulosclerosis, dilation of tubules,widening of interstitium, and cystic formation (Figs. 3, B–D,and 4). CS-3150 dose-dependently prevented both renal hy-pertrophy and histopathological changes.mRNA Expression in the Kidney. In the kidney of

DOCA rats, mRNA expression levels of profibrotic markers,such as TGF-b1 and Col1a1, and proinflammatory cytokines,such as IL-6 and MCP-1, were markedly increased comparedwith the control rats (Table 1). NADPH oxidase is known tocause reactive oxygen species production, and the mRNAexpression levels of NADPH oxidase subunits, such as p47phoxand p67phox, were also increased in the kidney of DOCArats (Table 1). Treatment with CS-3150 clearly inhibitedthe induction of mRNA expression of these markers in adose-dependent manner.

Experiment 2

SBP and UP Excretion. Consistent with experiment 1,SBP in the vehicle group was significantly higher than that inthe control group at 11 weeks of age, and additional 4-weekadministration of DOCA induced further SBP elevation (Fig.

5A). Under continuous DOCA treatment, CS-3150 inhibitedfurther SBP elevation at 13 weeks of age, but SBP at 15 weeksof age was equivalent to that in the vehicle group (Fig. 5A).Withdrawal of DOCA treatment did not show any significantdifference in SBPs at either 13 or 15 weeks of age. CS-3150treatment without DOCA administration dramatically low-ered SBP to the same level as the control group.UP excretion was markedly increased at 11 weeks of age,

and additional 4-week administration of DOCA caused afurther increase in UP excretion (Fig. 5B). CS-3150 signifi-cantly reduced UP excretion under continuous DOCA treat-ment (Fig. 5B). Withdrawal of DOCA treatment graduallydecreased UP excretion, and CS-3150 treatment withoutDOCA administration elicited a further reduction in UPexcretion (Fig. 5B).Other Urinary Parameters. UV was increased in the

vehicle group, and was gradually decreased by CS-3150treatment and/or withdrawal of DOCA treatment (Supple-mental Fig. 2A). CS-3150 treatment and/or withdrawal ofDOCA treatment did not show any significant effect onurinary Na1 and K1 excretions (Supplemental Fig. 2,B and C).At 15weeks of age, urinaryMCP-1 excretion was reduced by

CS-3150 treatment and/or withdrawal of DOCA treatment,compared with the vehicle group (Supplemental Table 2).Kidney Weight and Histology. In the vehicle group,

kidney weight was significantly increased compared with thecontrol group at 11 weeks of age, and DOCA treatment for afurther 4 weeks maintained this renal hypertrophy until15 weeks of age (Fig. 6A). Withdrawal of DOCA treatmentreduced kidney weight to the control level, meaning that MRactivation contributes to renal hypertrophy in this model. Infact, treatment with CS-3150 markedly reduced kidneyweight regardless of DOCA treatment.Consistent with experiment 1, marked histopathological

changes in kidney were observed in the vehicle group at11weeks of age, and further treatment withDOCA for 4 weeksexacerbated these changes, especially glomerulosclerosis

Fig. 2. Effects of CS-3150 (CS) on systolic blood pressure and urinary protein excretion in DOCA/salt-induced hypertensive rats (in experiment 1). From7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt (4% NaCl) diet. CS-3150(0.3–3mg/kg) was orally administered once a day for 4 weeks from the start date of DOCA administration. (A) Systolic blood pressure wasmeasured at 7,9, and 11 weeks of age. (B) Urine was collected for 24 hours at 11 weeks of age, and urinary volume and protein concentration were measured. Urinaryprotein excretion for 24 hours was calculated. Con, control group (no DOCA administered); CS0.3, 0.3 mg/kg CS-3150–treated group; CS1, 1 mg/kgCS-3150–treated group; CS3, 3 mg/kg CS-3150–treated group; Veh, vehicle-treated group. Data are expressed as the mean 6 S.E.M. (N = 6 in eachgroup). ##P , 0.01 versus control; *P , 0.05 versus vehicle; **P , 0.01 versus vehicle.

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(Figs. 6, B–D, and 7). Interestingly, under continuous treat-ment of DOCA, CS-3150 not only inhibited these changes butalso clearly ameliorated them, indicating that CS-3150 in-duced regression of renal injury.Withdrawal of DOCA treatmentalso resulted in a similar amelioration, and CS-3150 treatmentwithout DOCA administration elicited further improvement ofrenal histology.

mRNA Expression in the Kidney. Consistent withexperiment 1, mRNA expression levels of profibrotic markers(TGF-b1 and Col1a1), proinflammatory cytokines (IL-6 andMCP-1), and NADPH oxidase subunits (p47phox and p67phox)were markedly increased in the kidney of DOCA rats comparedwith the control rats at 11 weeks of age, and the same increasewas noted at 15weeks of age (Table 2). Under continuous DOCA

Fig. 4. Representative photomicrographs showing glomerulosclerosis and tubular injury in DOCA/salt-induced hypertensive rats (in experiment 1). CS,CS-3150.

Fig. 3. Effects of CS-3150 on kidney weight and histopathological changes in the kidney of DOCA/salt-induced hypertensive rats (in experiment 1).From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt (4% NaCl) diet.CS-3150 (0.3–3 mg/kg) was orally administered once a day for 4 weeks from the start date of DOCA administration. (A) At 11 weeks of age, the rightkidney was removed under anesthesia and weighed. The kidney weight/body weight (BW) ratio was calculated. (B–D) The sections of excised rightkidney were stained with periodic acid methenamine silver, and severity of glomerulosclerosis and tubular injury (cortex and medulla) wassemiquantitatively evaluated. Con, control group (no DOCA administered);CS0.3, 0.3 mg/kg CS-3150–treated group; CS1, 1 mg/kg CS-3150–treatedgroup; CS3, 3 mg/kg CS-3150–treated group; Veh, vehicle-treated group. Data are expressed as the mean 6 S.E.M. (N = 6 in each group). ##P , 0.01versus control; **P , 0.01 versus vehicle.

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treatment, CS-3150 clearly reduced the mRNA expression ofsome of these markers. Withdrawal of DOCA treatment alsoshowed a similar effect, and CS-3150 treatment without DOCAadministration elicited a further decrease in mRNA expression.

DiscussionThis is the first report that shows a therapeutic effect

of CS-3150 as well as a preventive effect on hypertensionand renal injury in DOCA/salt-hypertensive rats. CS-3150(0.3–3 mg/kg) dose-dependently prevented the elevation ofSBP induced by DOCA/salt loading. CS-3150 also suppressedthe development of renal injury (i.e., proteinuria, renal hyper-trophy, and histopathological changes in glomeruli and tubule)with inhibition of gene expression related to fibrosis, inflam-mation, and oxidative stress. In addition, CS-3150 at 3 mg/kgclearly ameliorated existing renal injurywithout affecting SBP,even under continuous DOCA treatment, indicating thatCS-3150 has the potential to regress established renal injuryin DOCA rats. Moreover, CS-3150 in combination with with-drawal of DOCA treatment showed further therapeutic effecton renal injury with reduction in SBP. Therefore, these are thefirst results to demonstrate that CS-3150 elicited regression ofovert proteinuria with histologic restoration of glomerular andtubular damage in DOCA rats.In this study, repeated administration of CS-3150 for

4 weeks significantly inhibited the onset of hypertension inthe DOCA rats, which was consistent with the results ofour previous study for 2 weeks (Arai et al., 2015). SBP wasmeasured by the tail-cuff method, which is a little lessaccurate than the telemetry method. Nevertheless, this wouldnot be a major issue since the dose-dependent antihyperten-sive effect of CS-3150 was clearly observed. DOCA/salt load-ing also caused marked proteinuria, renal hypertrophy, andglomerular and tubular injury, which were significantlyprevented by treatment with CS-3150. Interestingly, signifi-cant suppression of renal injury in the absence of a bloodpressure–lowering effect was detected at 0.3 mg/kg CS-3150,suggesting that the renal protective effect of CS-3150 couldnot be explained simply by its antihypertensive action.Accumulating evidence has shown that aldosterone/MR sig-naling directly contributes to the pathogenesis of renaldamage such as tubulointerstitial fibrosis through collagenaccumulation (Sun et al., 2006; Diah et al., 2008), increasedexpression of proinflammatory cytokines in the kidney (Blasiet al., 2003; Ikeda et al., 2009), and renal reactive oxygenspecies generation by an NADPH oxidase–dependent mecha-nism (Beswick et al., 2001; Nishiyama et al., 2004). In thecurrent study, upregulation of mRNA for several markers

related to fibrosis (TGF-b1 and Col1a1), inflammation (MCP-1and IL-6), and oxidative stress (p47phox and p67phox) wasobserved in the kidney of DOCA rats, and CS-3150 treatmentinhibited these changes. We also confirmed that increase inurinary MCP-1 excretion was inhibited by CS-3150 treatment(Supplemental Table 1). Taken together, the preventive effectof CS-3150 on renal injury could be due to not only antihy-pertensive but also direct renal protective effects throughantifibrotic, anti-inflammatory, and antioxidative actions.So far, renal injury such as glomerulosclerosis and tubular

injury has been considered to be irreversible once established(Klahr, 1999; Phillips et al., 1999). However, recent clinicaland nonclinical studies have shown that it can be reversed(Adamczak et al., 2003; Gaede et al., 2004; Rossing et al., 2005;Teles et al., 2009). In fact, regression of renal injury has beenreported to be achieved by intensive intervention against thecauses of renal injury, such as hypertension, diabetes, andhyperlipidemia (Hovind et al., 2001; Fioretto et al., 2006; Zojaet al., 2010). Since renal injury in DOCA rats is caused mainlyby MR activation, CS-3150 treatment in DOCA rats withestablished renal injury could mimic the intensive inter-vention described earlier. Surprisingly, CS-3150 elicited amarked reduction in proteinuria and kidney weight, anddramatically reversed glomerulosclerosis and tubular injurywith only a slight effect on blood pressure, indicating thatCS-3150 treatment restored the established renal damage byDOCA/salt loading independent of its antihypertensive action.There is some speculation about the mechanisms for this rever-sal of renal injury. It has been reported that degradation ofextracellular matrix protein would be directly involved in theregression of sclerosis lesion in the kidney by an angiotensin-converting enzyme inhibitor (Remuzzi et al., 2006) or angiotensinreceptor blocker (Boffa et al., 2003). In these reports, a decreasein expression of plasminogen activator inhibitor-1 (PAI-1), whichis known as an inhibitor of matrix degradation (Ma et al., 2000),was observed in the kidney, resulting in increased metallopro-teinase activity anddecreased expression of TGF-b1and collagentypes I and IV. In the present study, increasedmRNAexpressionof TGF-b1 and Col1a1 in the kidney was clearly downregulatedby CS-3150 treatment. Thus, in the kidney of DOCA rats,degradation of once accumulated extracellular matrix proteinmay occur, at least in part, through inhibition of PAI-1 byCS-3150 treatment. Brown et al. (2000) described the involve-ment of PAI-1 in aldosterone-induced renal injury, which couldalso support this hypothesis. Several reports have recentlysuggested that an injured kidney attempts to repair and re-generate in both its glomerular and tubular compartments, andstem/progenitor cells could contribute to this process throughmigration of adjacent undamaged cells (Choi et al., 2009; Benigni

TABLE 1Effects of CS-3150 on mRNA expression levels in the kidney in DOCA/salt-induced hypertensive rats (in experiment 1)Data are relative mRNA levels of each gene calculated by normalizing them to the peptidylprolyl isomerase B mRNA level, and are expressed asthe mean 6 S.E.M. (N = 6 in each group).

Group TGF-b1 Col1a1 IL-6 MCP-1 p47phox p66phox

Control 0.52 6 0.03 0.42 6 0.02 0.22 6 0.02 0.53 6 0.02 0.52 6 0.04 0.56 6 0.01Vehicle 1.40 6 0.13## 2.39 6 0.48## 2.54 6 0.39## 1.69 6 0.13## 1.46 6 0.18## 1.55 6 0.18##

CS 0.3 mg/kg 1.31 6 0.10 1.44 6 0.16* 1.77 6 0.17 1.43 6 0.11 1.16 6 0.09 1.31 6 0.13CS 1 mg/kg 0.75 6 0.06** 0.58 6 0.09** 0.59 6 0.12** 0.74 6 0.09** 0.74 6 0.08** 0.67 6 0.05**CS 3 mg/kg 0.54 6 0.01** 0.38 6 0.02** 0.21 6 0.04** 0.55 6 0.08** 0.56 6 0.02** 0.58 6 0.03**

CS, CS-3150.##P , 0.01 versus control; *P , 0.05 versus vehicle; **P , 0.01 versus vehicle.

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et al., 2010; Romagnani et al., 2013). For example, it has beenshown that progenitor-like cells are localized in the proximal anddistal tubule and migrate to the damaged lesion followingischemia (Maeshima et al., 2003). It has also been reported thattransplantation of bone marrow cells improves renal function byreplacement of defective podocytes inmicewith Alport syndrome(Prodromidi et al., 2006). Although the precise involvement ofthese cells remains unclear in the present study, CS-3150 may

enhance the renal tissue repair process through direct abolishingof renal MR activation by an exogenous DOCA. Specific exper-iments would be necessary to clarify this speculation.Additionally, CS-3150 treatment in combination with DOCA

withdrawal resulted in further regression of renal injury accom-panied by SBP reduction. Besides the classic role of MR in thekidney for blood pressure control, it has been shown thatextrarenal MR (e.g., brain and vasculature) is involved in the

Fig. 5. Effects of CS-3150 on systolic blood pressure and urinary protein excretion in DOCA/salt-induced hypertensive rats (in experiment 2). From7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt (4% NaCl) diet. From11 weeks of age, CS-3150 (CS; 3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. (A) Systolicblood pressure was measured at 7, 11, 13, and 15 weeks of age. (B) Urine was collected for 24 hours at 7, 11, 13, and 15 weeks of age, and urinary volumeand protein concentration were measured. Urinary protein excretion for 24 hours was calculated. Data are expressed as the mean 6 S.E.M. (N = 6 ineach group). ##P , 0.01 versus control; * P , 0.05 versus vehicle; **P , 0.01 versus vehicle.

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regulation of blood pressure (Oki et al., 2012;Barrett et al., 2013).For example, Rahmouni et al. (2002) reported that intracere-broventricular administration of an MR antagonist decreasedSBP in DOCA rats with developed hypertension. Experimentalstudies using transgenic mouse models, such as smooth musclecell–specific MR knockout mice (McCurley et al., 2012) andendothelial MR–overexpressingmice (Rickard et al., 2014), havesupported the direct involvement of vascular MR in bloodpressure control. From these findings, we speculate that notonly renal but also extrarenal MR activation in DOCA ratscould be completely inhibited by the combination of CS-3150treatment and withdrawal of DOCA, resulting in improvementof both renal injury and hypertension to the same level ascontrol rats. Additional experiments would be needed to testthis hypothesis.

Induction of urinary Na1 excretion (i.e., diuretic action)is known as a mechanism-based action of MR antagonists(Muller et al., 2003). However, several reports have suggestedthat it is not easy to assess the diuretic action of MRantagonists, especially in high-salt-induced hypertensionmodel rats (Chabert et al., 1984; Jiménez et al., 1988). Indeed,an increase in urinary Na1 excretion was not noted inCS-3150–treated DOCA rats in the current study, althoughthe precise cause remains unclear (Supplemental Figs. 1 and2). Also, the result of UV was well correlated with those ofSBP and UP excretion, and we think that this simply re-flects the inhibitory effects of CS-3150 on DOCA/salt-inducedpathology.Recently, Rafiq et al. (2014) demonstrated that high-salt

loading to Dahl salt-sensitive hypertensive rats induced renal

Fig. 6. Effects of CS-3150 (CS) on kidney weight and histopathological changes in the kidney of DOCA/salt-induced hypertensive rats (in experiment 2).From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt (4% NaCl) diet. From11 weeks of age, CS-3150 (3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. (A) At 11 or15 weeks of age, the right kidney was removed under anesthesia and weighed. The kidney weight/body weight (BW) ratio was calculated. (B–D) Thesections of excised right kidney were stained with periodic acid methenamine silver, and severity of glomerulosclerosis and tubular injury (cortex andmedulla) was semiquantitatively evaluated. Data are expressed as the mean6 S.E.M. (N = 6 in each group). $$P, 0.01 versus control at 11 weeks of age;##P , 0.01 versus control at 15 weeks of age; bbP , 0.01 versus vehicle at 11 weeks of age; **P , 0.01 versus vehicle at 15 weeks of age.

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injury that was reversed by switching to a normal salt diet(Rafiq et al., 2014). In another report, it has been shown that,in an adenine-loaded rat, which is widely used as a model for

renal failure, cessation of the adenine diet led to the reversal ofrenal damage (Shuvy et al., 2011). In the present study, inDOCA rats with established renal injury, withdrawal of

TABLE 2Effects of CS-3150 on mRNA expression levels in the kidney in DOCA/salt-induced hypertensive rats (in experiment 2)Data are relativemRNA levels of each gene calculated by normalizing them to the peptidylprolyl isomerase BmRNA level, and are expressed as themean6S.E.M. (N = 6 in each group).

Group TGF-b1 Col1a1 IL-6 MCP-1 p47phox p66phox

11 weeks of ageControl 0.47 6 0.02 0.47 6 0.05 0.15 6 0.02 0.43 6 0.03 0.46 6 0.05 0.47 6 0.02Vehicle 1.46 6 0.07$$ 2.54 6 0.24$$ 3.71 6 0.46$$ 1.61 6 0.05$$ 1.86 6 0.10$$ 1.50 6 0.08$$

15 weeks of ageControl 0.63 6 0.03 0.43 6 0.04 0.21 6 0.04 0.56 6 0.05 0.48 6 0.04 0.49 6 0.04Vehicle 1.64 6 0.07## 2.79 6 0.31## 1.63 6 0.28## 1.58 6 0.07## 1.09 6 0.14## 1.97 6 0.14##

CS 3 mg/kg 1.14 6 0.08**,b 1.01 6 0.08**,bb 0.97 6 0.11bb 1.32 6 0.08b 0.94 6 0.07bb 0.93 6 0.06**,bb

Vehicle w/o DOCA 1.10 6 0.07**,bb 0.97 6 0.12 **,bb 0.75 6 0.11*,bb 1.12 6 0.07**,bb 0.96 6 0.10bb 1.04 6 0.09**,bb

CS 3 mg/kg w/o DOCA 0.86 6 0.04**,bb 0.75 6 0.06**,bb 0.43 6 0.05**,bb 0.82 6 0.06**,bb 0.66 6 0.07bb 0.68 6 0.04**,bb

CS, CS-3150.$$P , 0.01 versus control (11 weeks of age); ##P , 0.01 versus control (15 weeks of age); bP , 0.05; bbP , 0.01 versus vehicle (11 weeks of age); *P , 0.05 versus vehicle;

**P , 0.01 versus vehicle (15 weeks of age).

Fig. 7. Representative photomicrographs showing glomerulosclerosis and tubular injury in DOCA/salt-induced hypertensive rats (in experiment 2). CS,CS-3150.

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DOCA administration itself reversed renal injury. Theseobservations suggest that elimination of a major contributorto the pathology (i.e., excess salt intake, adenine diet, orDOCA treatment) could lead to regression of renal injury.Although multiple causes are intricately involved in the path-ogenesis of renal injury in a clinical setting (Macisaac et al.,2014; Drawz and Rahman, 2015), these findings support theconcept that identification of the main pathways involved inkidney injury and targeted intensive treatment may lead toregression of renal injury.In conclusion, CS-3150 not only prevented the onset of

hypertension and renal injury, but also reversed them follow-ing their establishment in DOCA rats. In particular, to thebest of our knowledge, this is the first investigation on theregression of renal injury in DOCA rats, and CS-3150 clearlyexerted a regressive effect on overt proteinuria accompaniedby histologic restoration of glomerular and tubular dam-age. Therefore, CS-3150 could be a promising agent for thetreatment of hypertension and renal disorders, and may havepotential as a therapeutic approach to renal injury.

Acknowledgments

The authors thank Shin Nippon Biomedical Laboratories, Ltd.,Drug Safety Research Laboratories (Kagoshima, Japan) for theirexpert experiments.

Authorship Contributions

Participated in research design: Arai, Morikawa, Ubukata,Homma.

Conducted experiments: Arai, Morikawa, Ubukata, Homma.Contributed new reagents or analytic tools: Tsuruoka.Performed data analysis: Arai, Morikawa, Ubukata, Homma.Wrote or contributed to the writing of the manuscript: Arai,

Homma.

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Address correspondence to: Kiyoshi Arai, End-Organ Disease Laboratories,Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710,Japan. E-mail: arai.kiyoshi.ch@daiichisankyo.co.jp

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