1521-01033633402ndash410$2500 httpsdoiorg101124jpet117244459THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 363402ndash410 December 2017Copyright ordf 2017 by The American Society for Pharmacology and Experimental Therapeutics

Sustained Activation of Guanylate Cyclase-A with TDT aNatriuretic Peptide Derivative Exhibits CardiorenalProtection in Dahl Salt-Sensitive Hypertensive Ratss

Shohei Oishi Naoko Suzuki Yuri Hasui Tsuyoshi Homma Masanori ObanaTakahiro Nagayama and Yasushi FujioLaboratory of Clinical Science and Biomedicine Graduate School of Pharmaceutical Sciences Osaka University Osaka Japan(SO MO YF) End-Organ Disease Laboratories (SO YH TH) Rare Disease amp LCM Laboratories (TN) and ResearchFunction (NS) Daiichi Sankyo Co Ltd Tokyo Japan

Received August 3 2017 accepted October 2 2017

ABSTRACTHeart failure often presents with prognosis-relevant impairedrenal function To investigate whether the chronic activation ofguanylate cyclase-A (GC-A) protects both heart and kidneywe examined the effects of TDT a neprilysin (NEP)-resistantnatriuretic peptide (NP) derivative on cardiac and renal dysfunc-tion in Dahl salt-sensitive hypertensive (DS) rats Pretreatmentwith NEP or NEP inhibitor did not influence GC-A activation byTDT both in vitro and in vivo resulting in a long-acting profile ofTDT compared with native human atrial NP (hANP) The repeatedadministration of TDT to DS rats suppressed the progress ofcardiac hypertrophy systolicdiastolic dysfunction and pro-teinuria in a dose-dependent manner Compared with vehicleand hANP salt dietndashinduced podocyte injury was reduced byTDT as analyzed by urinary podocalyxin concentration renal

expression of nephrin mRNA and glomerular expression ofdesmin protein Since glomerular TRPC6 plays detrimental rolesin podocyte homeostasis we examined the renal expression ofTRPC6 in DS rats and found that salt diet upregulated theexpression of TRPC6 Importantly TRPC6 induction was signif-icantly decreased in TDT-treated rats compared with vehicleand hANP Consistently in primary-culture podocytes from DSrats TDT inhibited ATP-induced calcium influx similar to TRPCinhibitor SKF96365 Finally TDT-mediated protection of podo-cytes was abolished by protein kinase G inhibitor KT5823 Inconclusion TDT treatment attenuated heart and kidney dys-function accompanied by podocyte protection through inhibi-tion of TRPC6 Thus long-acting NPs could be a new avenue fortreatment of heart failure

IntroductionApproximately 26 million people worldwide suffer from

heart failure (HF) which is one of the major causes of death(Ambrosy et al 2014) Blockade of neurohumoral systemssuch as the renin-angiotensin-aldosterone system (RAAS)and the sympathetic nervous system (SNS) improves theprognosis of patients with HF significantly but its efficacyis still unsatisfactory Patients with HF especially thosecaused by hypertension have a high prevalence of prognosis-relevant comorbidities such as chronic kidney diseaseAccumulating evidence has demonstrated that interactionbetween the heart and kidney bidirectionally acceleratesthe progression of their pathologies which are termedcollectively as cardiorenal syndrome (Schefold et al 2016)Therefore therapeutic strategies against HF should have astheir objective not simply cardioprotection but alsorenoprotection

Natriuretic peptides (NPs) including atrial natriureticpeptide (ANP) and B-type natriuretic peptide bind to theirreceptor guanylate cyclase-A (GC-A) thereby enhancingintracellular cGMP followed by activation of protein kinaseG (PKG) The NPGC-AcGMPPKG cascade functions as a keyregulator of body fluid and blood pressures regulatingmultiplephysiologic outputs such as natriuresis vasodilation andrenin-angiotensin-aldosterone inhibition and it has been pro-posed that NPs play protective roles in cardiac and renaldysfunction (Volpe 2014) In this context carperitide recombi-nant human ANP is clinically used as a primary medication totreat acute heart failure (AHF) patients in Japan Moreover onthe basis of the evidence from preclinical experiments usinggenetically engineered animals (Kishimoto et al 2009) andfrom population-based human studies (Seidelmann et al2017) it has been theorized that chronic enhancement of bloodNPs could improve prognosis in chronic heart failure (CHF)patients However in spite of the beneficial effects of treatmentwith NPs chronic treatment of native NPs is not practicalbecause NPs are enzymatically degraded by neprilysin (NEP)and have short half-lives (Hashimoto et al 1994)

httpsdoiorg101124jpet117244459s This article has supplemental material available at jpetaspetjournalsorg

ABBREVIATIONS ANP atrial natriuretic peptide CHF chronic heart failure CHO Chinese hamster ovary DS Dahl salt-sensitive hypertensiveDWS diastolic wall strain GC-A guanylate cyclase-A HF heart failure HR heart rate KT5823 PKG inhibitor VII NEP neprilysin NGAL neutrophilgelatinase-associated lipocalin PKG protein kinase G SBP systolic blood pressure SKF96365 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole UP urinary protein

402

httpjpetaspetjournalsorgcontentsuppl20171011jpet117244459DC1Supplemental material to this article can be found at

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Recent peptide engineering technology has structurallymodified native NPs to prolong their half-lives by conferringresistance to enzymatic degradation by NEP (Meems andBurnett 2016) In this study TDT an NEP-resistant de-rivative of taipan natriuretic peptide (TNP) (Alewood et al2008) was used to achieve sustained activation of GC-AConsidering that NEP inhibitor in combination with angio-tensin II receptor blocker clinically improves the prognosis ofCHF patients without worsening renal function (McMurrayet al 2014) it is probable that TDT could be a novel promisingdrug for CHF however its pharmacological characterizationand medical benefits remain to be elucidatedHere we have hypothesized that sustained activation of

GC-A could improve cardiac and renal disorders of HF thatresult from hypertension And we examined the effects ofsustained GC-A activation by TDT on cardiac and renaldysfunction using Dahl salt-sensitive hypertensive (DS) rats

Materials and MethodsPeptides TDT (SDSKIGNGCFGHKIDRINHVSNLGCNRIMQNPP-

KKFSGE disulfide bond between C9-C25) was synthesized by Scrum Inc(Tokyo Japan) as described in patent no US2008015374 (Alewood et al2008) The purity was confirmed by high-performance liquid chromatog-raphyanalysis to be95hANPwaspurchased fromDaiichiSankyoCoLtd (Tokyo Japan)

Animals All experimental procedures were approved by theInstitutional Animal Care and Use Committee of Daiichi SankyoCo Ltd The investigation conformed to the Guide for the Care andUse of Laboratory Animals 8th ed updated by the US NationalResearch Council Committee in 2011

In Vitro Human GC-AcGMP Assay The open reading frame ofhuman GC-A (hGC-A) constructed in vector pCMV6-Entry (cat noRC209267) was purchased from OriGene Technologies Inc (RockvilleMD) Chinese hamster ovary (CHO)-K1 cells were stably transfectedwith the plasmid DNA by Lipofectamine (Thermo Fisher ScientificWaltham MA) and selected by G418 (Thermo Fisher Scientific) using alimiting-dilution method to obtain a single clone CHOhGC-A stableclones were then screened for the best response to hANP

For the cGMP assay hGC-A-expressing or parental CHO cells wereseeded in a 384-well plate at 4 103 cellswell and cultured for 1 dayunder 5 CO2 at 37degC After removal of culture medium the cells wereincubated with 16 mM 1-methyl-3-isobutylxanthine (Merck MilliporeBillericaMA) for 10minutes followed by incubation for 15minuteswithhANP or TDTwhich had been preincubatedwith orwithout 10mgml ofNEP (RampDSystemsMinneapolisMN) for 30minutes at 37degC The cellswere lysed and then cGMP concentration in the lysate was determinedusing a cGMP assay kit (Cisbio Codolet France)

In Vivo Plasma cGMP Measurement Plasma cGMP concen-tration was evaluated using 8-week-oldmale SD rats under isoflurane(2) anesthesia hANP or TDT at 100 nmolkg was administrated viabolus injection just after intravenous infusion of saline or phosphor-amidon (Santa Cruz Biotechnology Dallas TX) an NEP inhibitor at825 nmolkg per minute for 2 minutes followed by continuousadministration at 165 nmolkg per minute until the end of theexperiment according to a previous report (Hashimoto et al 1994)cGMP concentration in Na2EDTA plasma at each time point wasmeasured by Amersham cGMP Enzymeimmunoassay Biotrak (EIA)System (GE Healthcare Little Chalfont UK)

Experimental Protocol of Chronic Study Male DS rats werepurchased from Japan SLC Inc (Shizuoka Japan) The rats weremaintained in a room at 50 relative humidity (acceptable range30ndash70) and 22degC (acceptable range 19ndash25degC) under a 12-hourlightdark cycle and allowed free access to diet (FR-2 FunabashiFarm Chiba Japan) and water

At 7 weeks of age rats began a high-salt diet (FR-2 containing 8NaCl Funabashi Farm) and at 12 weeks of age were divided into fourgroups (n5 13ndash14) on the basis of body weight systolic blood pressure(SBP) diastolic wall strain (DWS) left ventricular end-diastolicdimension and urinary protein (UP) excretion The vehicle (5glucose) hANP (40 nmolkg) or TDT (low dose 8 nmolkg or highdose 40 nmolkg) was subcutaneously administrated twice a day for8 weeks up to 20 weeks of age As a control six rats were fed a normal-salt diet (FR-2 containing 03 NaCl)

Measurement of Systolic Blood Pressure and Heart RateSBP and heart rate (HR) were measured by the tail-cuff method usinga noninvasive sphygmomanometer (BP-98A Softron Tokyo Japan)under conscious restrained conditions at 11 (before dosing) 16 and19 weeks of age Individual data are presented as the mean values ofthree consecutive measurements

Measurement of Urinary and Blood Biochemical Parame-ters Twenty-four-hour urine collection and blood sampling wereperformed at 11 and 18 weeks of age Urinary protein concentrationand plasma creatinine concentration were measured by an automatedclinical analyzer (BiOLiS 24i Premium Tokyo Boeki MachineryTokyo Japan) and urinary electrolytes were detected by an electrolyteanalyzer (STAX-2 Techno Medica Kanagawa Japan) Urinarypodocalyxin (Excocell Philadelphia PA) and kidney injury molecule-1 (KIM-1 RampD systems) were determined by ELISA kits

Echocardiography High-resolution transthoracic echocardiog-raphy was performed using Vevo 2100 equipped with a 13-MHz lineararray transducer (Fujifilm Visualsonics Toronto ON Canada) underisoflurane (15ndash3) anesthesia at 11 and 19 weeks of age as describedpreviously (Kumagai et al 2016) Administration of test peptides wasstopped on theday of themeasurementAccording to themethod ofTakedaet al (2009) DWS was calculated as an indicator of myocardial wallstiffness using the parameters of LVposteriorwall thickness at end-systole(LVPWs) and at end-diastole (LVPWd) using the following equation

DWS5 12LVPWs=LVPWd

Hemodynamic Measurements The hemodynamic study wasperformed at 20 weeks of age under 2 isoflurane anesthesia using acatheter pressure transducer (Millar Instruments Houston TX)according to a previously described method (Klotz et al 2006) The

Fig 1 TDT induces cGMP production via GC-A in an NEP-resistantmanner CHO cells stably expressing human GC-A were incubated withthe indicated concentrations of peptides for 15 minutes after preincuba-tion with or without NEP for 30 minutes cGMP concentrations weremeasured with a Cisbio HTRF-based assay Representative data areshown from three independent experiments that were performed inquadruplicate Data are presented as the mean 6 SEM Error bars thatdo not show are smaller than symbols

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time constant of LV isovolumic pressure decline tau was calculatedusing the Glantz method (Raff and Glantz 1981)

Histopathological Evaluation The heart samples fixed in 10neutral buffered-formaldehyde solution were embedded in paraffinSections were stained with hematoxylin and eosin Cross-sectionalareas of cardiomyocytes were measured by a researcher who wasblinded to the experimental conditions as the mean value of100 eosin-positive cardiomyocytes of the papillary muscle usingmorphometric analysis software (analySIS Soft Imaging SystemMuumlnster Germany) in a randomly selected microscope field in eachsection

Quantitative PCR Relative gene expression in the kidney wasdetermined by quantitative real-time polymerase chain reaction(PCR) using TaqMan Gene Expression Assays and TaqMan universalPCR master mix (Thermo Fisher Scientific) according to a generalprocedure (Arai et al 2016) The following assays were used B cellleukemialymphoma 2 (Bcl2 Rn99999125_m1) collagen 1a1 (Col1a1Rn01463848_m1) interleukin-1b (IL-1b Rn00580432_m1) nephrin(Nphs1 Rn00674268_m1) neutrophil gelatinase-associated lipocalin(NGAL Lcn2 Rn00590612_m1) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH Rn99999916_s1) PCR reactions were per-formed using a 7900 HT Fast Real Time PCR system (Thermo Fisher

Fig 3 Effects of chronic TDT therapy on survival body weight systolic blood pressure and heart rate in DS rats DS rats were fed a high-salt diet (8NaCl) from 7 weeks of age hANP or TDT was twice-daily subcutaneously dosed from 12 weeks of age for 8 weeks Control normal diet (03 NaCl)-fedgroup (n = 6) Vehicle vehicle-treated group (n = 14) hANP hANP (40 nmolkg)-treated group (n = 14) TDT_L TDT (8 nmolkg)-treated group (n = 13)TDT_H TDT (40 nmolkg)-treated group (n = 13) (A) Survival rate (B) Body weight (C) SBP (D) HR Data are presented as the mean 6 SEM Errorbars that do not show are smaller than symbols daggerP 005 DaggerP 001 vs control P 005 P 001 vs vehicle

Fig 2 TDT administration enhancesplasma cGMP concentration independentlyof NEP activity in rats (A) After bolusinjection of TDT or hANP at 100 nmolkg inthe presence or absence of phosphoramidonan NEP inhibitor (NEPi) plasma concentra-tions of cGMP were measured (B) AUC of thechange in plasma cGMP concentration frombaseline is shown Data are presented asthe mean6M SEM (n = 3 each) $P 005$$P 001 vs hANP-treated group

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Scientific) Relative mRNA levels of each gene were calculated bynormalizing them to GAPDH mRNA level

Glomerular Protein Expression The kidney samples fixed in10 neutral buffered-formaldehyde solution were embedded inparaffin and then sectioned Glomerular expression of TRPC6and desmin was evaluated by semiquantitative scoring of immuno-histostained samples Immunohistostaining was performed with anEnVision1 Kit (Agilent Santa Clara CA) in accordance with theprocedure described by the manufacturer Rabbit anti-rat TRPC6 (1100 dilution ACC-120 Alomone Laboratories Jerusalem Israel) andmouse anti-rat desmin (1400 dilution D33 Abcam Cambridge UK)antibodies were used as primary antibodies (Nijenhuis et al 2011Sonneveld et al 2013) Glomerular TRPC6 and desmin expressionwas scored semiquantitatively from 0 to 5 on the basis of the extent ofimmunostaining intensity in 20 randomly selected glomeruli perkidney (negative 5 0 1ndash20 positive with mild staining 5 1 1ndash20positive with intense staining or 21ndash40 positive with mild staining5 2 21ndash40 positive with intense staining or 41ndash60 positive withmild staining 5 3 41ndash60 positive with intense staining or 61ndash80positive with mild staining 5 4 and 61ndash100 positive with intensestaining 5 5) Scoring was performed independently by two investi-gators who were blinded to the experimental conditions

Primary Cultured Rat Podocytes Eighteen- to 21-week-old DSrats were fed an 8 NaCl-containing diet from 7 weeks of agePodocytes were isolated according to the conventional sieving method(Chen et al 2006) with minor modification In brief renal corticaltissue homogenates were strained through steel sieves with pore sizesin the order of 250 150 and then 75mm The glomeruli retained on thetop of the 75-mm sieve were washed and resuspended in ice-coldculturemedium (Dulbeccorsquos modified Eaglersquos mediumF-12 containing5 fetal bovine serum supplemented with 05 insulin-transferrin-selenium-A and 100 IUml penicillin-streptomycin) The glomeruliwere then plated on collagen I-coated 10-cm dishes After 4 daysrsquoculture in 5 CO2 at 37degC outgrowing glomerular cells were trypsi-nized and passed over a 40-mm nylon strainer Only cells passingthrough the strainer were replated on collagen I-coated plates andcultured for 1 day The cultured cells mostly consisted of arborized orcobble stone-like podocytes under the light microscope

Intracellular Calcium Measurements Cultured rat podocytesseeded at 3 104 cellswell in a 96-well plate for 1 day were washedtwice with phosphate-buffered saline and then suspended in RPMI1640 mediumwith or without 1 mMKT5823 (MerckMillipore) a PKGinhibitor or 10 mM SKF-96365 (Merck Millipore) a TRPC inhibitorNinety minutes after the incubation cells were treated with 10 mMFura 2-AM (Thermo Fisher Scientific) and incubated for 30 minutesfollowed by wash and treatment with 100 nM TDT dissolved in Krebs-Ringer phosphate buffer (150 mM NaCl 6 mM KCl 1 mM MgCl210 mM D-glucose and 10 mM HEPES) containing 15 mM CaCl2 for15 minutes The culture plates were mounted on an invertedmicroscope stage and exposed to 340- and 380-nm wavelength lightusing a multiple wavelengths high-solution fluorescence microscopysystem (IonOptixWestwoodMA) The arborized podocytes were thenselected and monitored To stimulate calcium entry into the cells10 mM ATP (Merck Millipore) was added Data were presented as thefluorescence ratio 340380 nm

Statistical Analysis Dataare shownasmeans6SEMStatisticalanalyses between two groups were performed by Studentrsquos or Welchrsquos ttest For multiple comparisons the Dunnettrsquos test or Tukeyrsquos test wasperformed depending on the purpose Survival analysis was performedusing Kaplan-Meier analysis and the log-rank test A value of P 005was considered to be statistically significant All statistical analyseswereperformed using SAS System Release 92 (SAS Institute Tokyo Japan)

ResultsTDT Activated GC-A in an NEP-Resistant Manner

To assess activation of GC-A by TDT we measured theconcentrations of cGMP in human GC-A (hGC-A)-expressing

cells after stimulation with TDT or hANP (Fig 1) TDTshowed a full agonistic activity to hGC-A relative to hANPin a concentration-dependent manner though the EC50 value

Fig 4 Chronic TDT therapy inhibited cardiac hypertrophy in DS ratsRats were fed a high-salt diet concomitant with treatment of hANPor TDT (A) The left ventricular weight (LV)body weight (BW) ratiowas measured (n = 6 for control n = 13ndash14 for the other groups)(B) Representative images of hematoxylin and eosinndashstained sections ofthe left ventricle are shown (scale bar 50 mm) (C) Cross-sectional areas ofcardiomyocytes were measured (n = 4 for control n = 8 for the othergroups) Data are presented as the mean 6 SEM DaggerP 001 vs controlP 005 P 001 vs vehicle

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of TDT was higher than that of hANP (TDT 195 pM hANP19 pM) Importantly preincubation of hANP with NEPreduced hGC-A activation by hANP but not that of TDTTo evaluate the activation of GC-Awith TDT in vivo TDT or

hANP was intravenously administered and plasma cGMPconcentrations were measured Plasma cGMP concentrationswere more potently elevated and more slowly reduced by TDTthan by hANP (Fig 2A) Consistent with in vitro experimentsNEP inhibition by phosphoramidon enhanced AUC of cGMPafter hANP administration up to a level similar to TDT alonebut did not significantly influence AUC after TDT treatment(Fig 2B)Chronic Treatment of TDT Ameliorated Salt-Induced

Hypertensive Heart Failure in DS Rats To address thebeneficial therapeutic properties of TDT DS rats were fed ahigh-salt diet and mortality body weight blood pressure andheart rate were analyzed Kaplan-Meier analyses revealedthat the salt diet reduced the survival rate in DS rats and thatmortality was improved by the treatment with TDT comparedwith vehicle (Fig 3A) In this experiment we used two dosagesof TDT and confirmed that TDT improved salt-inducedmortality in a dose-dependent manner Only one animal in14 animals in TDT at low dosage (TDT_L) group died throughthe experiment whereas 6 of 14 in vehicle group diedConsidering that TDT at high dosage (TDT_H) perfectlyimproved survival TDT_L seemed to be effective but statis-tical significance between TDT_L and vehicle groups was notobserved probably because animals died concentratedly from6 to 8 weeks in the vehicle group Consistent with theincreased mortality body weight gradually decreased invehicle-treated rats whereas body weight increased in TDT-treated animals with a significant difference at week 6 (Fig3B) SBP was significantly elevated by the high-salt diet atthe initiation of treatment with NPs compared with controlSubsequent salt loading continued to elevate SBP compara-bly in vehicle hANP and TDT_L group which was sup-pressed by TDT_H at week 4 (Fig 3C) Compared with thecontrol the high-salt diet increased HR in the vehicle-treated group but TDT significantly reduced HR at week6 (Fig 3D)The high-salt diet induced cardiac hypertrophy with an

increase of the left ventriclendashtondashbody weight (LVBW) ratiowhich was reduced by TDT in a dose-dependent manner(Fig 4A) Likewise histologic analyses demonstrated thatthe increase in cardiomyocyte size induced by the high-saltdiet was reduced by hANP and TDT (Fig 4B and 4C)

To examine the effects of TDT on cardiac function echocar-diographic analyses (Table 1) and hemodynamic assessments(Table 2) were performed Echocardiographic examinationdemonstrated that the high-salt diet reduced fractionalshortening ejection fraction stroke volume cardiac outputand DWS an indicator of ventricular wall stiffness and thatboth TDT_L and TDT_H attenuated the impairment of theseparameters Consistently hemodynamic analyses using acatheter showed that both systolic (dPdtmaxP) and diastolic(dPdtminP and Tau) functions were worsened by the high-saltdiet and that TDT ameliorated these functions Of notemeasurement by catheter method demonstrated that hANPand TDT_L did not lower BP whereas TDT_H significantlyreduced BP consistent with the data by tail cuff methodTDT Protected Renal Disorder Induced by a High-

Salt Diet in DS Rats Renal function was analyzed 6 weeksafter NP treatment (Table 3) UP excretion was increased inthe high-salt diet groups comparedwith the normal-diet groupat the initiation of NP therapy (data not shown) UP excretionprogressively increased 10 times in the vehicle groupcompared with the control group Importantly TDT sup-pressed the increase in UP excretion induced by the high-salt diet Although the sodium excretionintake ratio was notinfluenced by NP treatment each peptide potently enhancedcreatinine clearance with statistical significance comparedwith the vehicle-treated group To support these findings wedetermined renal mRNA expression of inflammatory cytokineIL-1b profibrotic marker collagen 1a1 and antiapoptoticmarker Bcl-2 Salt overload upregulated IL-1b and collagen1a1 and downregulated Bcl-2 whereas hANP and TDTsignificantly inhibited these changes (Supplemental Figure)Podocyte and Tubular Injury Were Suppressed by

TDT Given the antiproteinuric effect of TDT we nextevaluated injury markers of tubules and podocytes whichplay a pivotal role in the glomerular filtration barrier UrinaryKim-1 excretion andmRNA expression of NGAL in the kidneywere attenuated by TDT in a dose-dependent manner in-dicating that hANP and TDT ameliorated tubular injury(Fig 5A andB) Urinary excretion of podocalyxin a componentof the slit diaphragm in podocytes was remarkably higher inthe vehicle-treated group than in the control group whereasboth TDT_L and TDT_H but not hANP significantly sup-pressed the elevation (Fig 5C) Consistently mRNA expres-sion of nephrin an important component of the renal filtrationbarrier was upregulated by TDT in a dose-dependent manner(Fig 5D) but not by hANP Consistent with these results

TABLE 1TDT preserved cardiac systolic and diastolic function in DS ratsCardiac systolicdiastolic functional parameters were measured by echocardiography at 19 weeks of age (n = 6 for controln = 11 for vehicle and hANP n = 12 for TDT_L and n = 13 for TDT_H) Data are presented as the mean 6 SEMdaggerP 005 DaggerP 001 vs control group P 005 P 001 vs vehicle-treated group

Control Vehicle hANP TDT_L TDT_H

LVDd (mm) 750 6 024 773 6 031 788 6 016 769 6 021 784 6 020IVSd (mm) 216 6 013 284 6 008Dagger 259 6 008 261 6 008 235 6 010LVPWd (mm) 207 6 009 262 6 010Dagger 245 6 005 255 6 007 232 6 005FS () 410 6 12 292 6 33dagger 349 6 20 404 6 25 448 6 17EF () 701 6 15 530 6 46dagger 619 6 29 686 6 30 740 6 19SV (ml) 210 6 14 161 6 6 Dagger 204 6 8 214 6 10 244 6 13CO (mlmin) 849 6 53 624 6 30Dagger 787 6 31 816 6 42 932 6 49DWS 0328 6 0032 0191 6 0021Dagger 0262 6 0018 0272 6 0017 0348 6 0011

CO cardiac output EF ejection fraction FS fractional shortening IVSd intraventricular septum in diastole LVDdleft ventricular diameter in diastole LVPWd left ventricular posterior wall in diastole SV stroke volume

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immunohistological analyses using anti-desmin antibodydemonstrated that glomerular desmin expression an estab-lished podocyte injury marker was less remarkable in theTDT-treated groups compared with the vehicle- and hANP-treated groups (Fig 5E and F) Taken together TDT amelio-rated both tubular and podocyte injury Since TDT morepotently protected from podocyte injury than did hANP and arecent report suggested that GC-A plays pivotal roles inpodocyte homeostasis we focused on the podocytesTDT Reduced Glomerular TRPC6 Expression in a

Dose-Dependent Manner To address the key renoprotec-tive mechanism of TDT we examined the glomerular proteinexpression level of TRPC6 since the upregulation of TRPC6expression results in the impairment of podocyte homeostasis(Moller et al 2007) As depicted in Fig 6 TDT reduced TRPC6expression elevated by salt overloading in a dose-dependentmanner Importantly TDT_L exhibited inhibitory effects onTRPC6 induction more remarkably than hANP suggestingthat TDT could be a promising renoprotective drugTo examine whether TDT directly transduces its signals in

podocytes we stimulated cultured rat podocyteswith TDT andmeasured cGMP production (Fig 7A) TDT stimulation pro-moted cGMP production suggesting that TDT could preservepodocyte homeostasis directly Since TRPC6-mediated cal-cium influx deteriorates podocyte homeostasis (Winn et al2005) we tested the effect of TDT on intracellular calciuminflux evoked by ATPwith or without SKF-96365 and KT5823in primary rat podocytes (Fig 7B and C) Robust calciuminflux induced by ATP was largely inhibited by pretreatmentwith SKF-96365 a TRPC inhibitor indicating that calciuminflux was largely mediated by TRPC TDT reduced calciuminflux andKT5823 a PKG inhibitor completely abolished theeffect of TDT suggesting that TDT regulated TRPC functionthrough PKG Importantly the treatment with SKF-96365 in

combination with TDT did not exert additional inhibitioncompared with SKF-96365 alone indicating that TDTinhibited calcium influx mainly by inhibiting TRPC

DiscussionHF is one of the major causes of death Since the NEP

inhibitor in combination with angiotensin receptor blockerimproves the prognosis of HF we hypothesized that NEP-resistant NPs could prevent HF progression In the presentstudy we examined the effects of TDT an NEP-resistant NPon cardiac and renal dysfunction in DS rats The repeateddosing of TDT ameliorated high-salt dietndashinduced cardiachypertrophy and suppressed systolic and diastolic dysfunc-tion In addition to cardioprotective effects TDT attenuatedproteinuria induced by the high-salt diet Moreover TDTsuppressed podocyte injury associated with the downregula-tion of TRPC6 expression Interestingly TDT directly acti-vated GC-A and reduced calcium influx by inactivatingTRPC6 activity in podocyte Thus we propose that there areprotective effects of chronic treatment with NEP-resistantNPs as a long-acting GC-A activator against cardiac and renaldisordersEntresto a combination drug of theNEP inhibitor sacubitril

and angiotensin receptor blocker valsartan is clinically usedas a novel anti-CHF drug to reduce the risk of cardiovasculardeath and hospitalization (McMurray et al 2014) Thecardioprotective effects of NEP inhibition are thought to beexplained largely by the elevation of endogenous NPs(Solomon et al 2012) However since NEP inactivates a widerange of peptide hormones and secretory factors in addition toNPs (Campbell 2017) it was difficult to make clear whetherexogenous NP treatment could ameliorate CHF Despiterecent advances in peptide engineering technology and

TABLE 3TDT improved renal function in DS ratsUrine collection for 24 hours was performed at 18 weeks of age Data are presented as the mean 6 SEM daggerP 005 DaggerP 001 vs control groupP 005 P 001 vs vehicle-treated group

Control (n = 6) Vehicle (n = 10) hANP (n = 11) TDT_L (n = 12) TDT_H (n = 13)

UV (mlday) 131 6 30 959 6 92Dagger 885 6 86 765 6 54 785 6 57Water intake (gday) 35 6 3 110 6 9Dagger 105 6 8 94 6 6 96 6 6UPday (mgday) 27 6 3 308 6 32Dagger 277 6 45 236 6 32 190 6 32Na+ excretionintake (gg) 058 6 009 031 6 001dagger 032 6 001 033 6 001 032 6 001CCr (mlmin) 20 6 04 24 6 03 35 6 04 36 6 03 42 6 03

CCr creatinine clearance UV urine volume

TABLE 2TDT improved hemodynamics in DS ratsHemodynamic parameters were measured by catheter with a pressure transducer at 20 weeks of age (n = 5 for control n = 7 for vehicle n = 6 forhANP n = 9 for TDT_L n = 9 for TDT_H) Data are presented as the mean 6 SEM daggerP 005 DaggerP 001 vs control group P 005 P 001vs vehicle-treated group

Control Vehicle hANP TDT_L TDT_H

SBP (mmHg) 138 6 4 236 6 3Dagger 226 6 5 227 6 7 206 6 6DBP (mmHg) 97 6 4 172 6 2Dagger 164 6 4 165 6 5 149 6 5dPdtmax (mmHgsec) 8099 6 402 10519 6 767 9987 6 451 12195 6 714 11144 6 625dPdtmaxP (sec) 987 6 30 782 6 52 dagger 825 6 25 929 6 41 939 6 19dPdtmin (mmHgsec) 29706 6 652 29073 6 582 28971 6 702 210917 6 511 211594 6 346dPdtminP (sec) 21011 6 44 2517 6 41Dagger 2596 6 42 2685 6 54 2769 6 36LVEDP (mmHg) 95 6 11 131 6 26 113 6 15 100 6 16 93 6 15Tau (msec) 120 6 06 248 6 38dagger 202 6 14 185 6 14 163 6 09

DBP diastolic blood pressure LVEDP left ventricular end diastolic pressure

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success in half-life extension of native NPs there have beenvery few reports to reveal the therapeutic potential of theirchronic application Here we focused on TDT as a novel NEP-

resistant NP TDT is an engineered NP derivative from taipannatriuretic peptide which is isolated from the venom of theinland taipan (Alewood et al 2008) Since TDT was resistant

Fig 5 TDT but not hANP protected tubules and podocytes from high salt-induced injury in DS rats (A and B) Tubular injury markers and (CndashF)podocyte injury markers were investigated (A) Urinary excretion of KIM-1 an injury marker of proximal tubules and (C) that of podocalyxin anindicator of podocyte detachment and fragmentation were measured at 18 weeks of age (n = 5 for control n = 10 for vehicle n = 11 for hANP n = 12 forTDT_L and n = 13 for TDT_H) (B) mRNA expression of NGAL another indicator of tubular injury and (D) that of nephrin a component of podocyteswere determined by real-time quantitative PCR in the DS rat kidneys at 20 weeks of age and normalized to the GAPDH mRNA level (n = 4 for controln = 8 for the other groups) (E and F) Glomerular protein expression of the podocyte injury marker desmin was evaluated by immunohistochemicalanalysis (E) Representative images are shown (scale bar 50 mm) (F) Desmin protein expression was semiquantitatively shown (n = 4 for controln = 8 for the other groups) Data are presented as the mean 6 SEM DaggerP 001 vs control P 005 P 001 vs vehicle or indicated group

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to degradation by NEP and exhibited cardiorenal protectiveeffects we could propose that the NEP-resistant NP would bea novel therapeutic strategy at least for HF patients in-tolerant of NEP inhibitors In addition considering that theupregulation of endogenous NPs by NEP inhibitor is theoret-ically limited exogenous NPs could additionally activateGC-A Further studies would be required to make clearwhether TDT shows clinical benefits additive to NEPinhibitorsRecent studies using genetically modified mice showed that

podocyte-specific ablation of the NP receptor GC-A generesults in increased susceptibility to renal injury (Staffelet al 2017) indicating that GC-A in podocytes play importantroles in renal protection however it remains to be fullyelucidated whether pharmacological activation of the NPreceptor protects kidneys from chronic stress because con-ventional NPs are rapidly degraded byNEP Here using TDTwe successfully demonstrated that chronic activation of GC-A

by long-acting NPs prevented renal dysfunction accompaniedby protection of podocytes Most importantly TDT protectedpodocytes even at a low dose that did not significantly impactblood pressure glomerular filtration rate and natriuresisConsidering the limitation of the dosing amount of GC-A

Fig 6 TDT reduced glomerular expression of TRPC6 protein in DS ratsGlomerular expression of TRPC6 protein in DS rats at 20 weeks of age wassemiquantitatively determined by immunohistochemical analysis (A) Rep-resentative images are shown (Scale bar 50 mm) (B) Glomerular expressionof TRPC6 protein was semiquantitatively shown (n = 4 for control n = 8 forthe other groups) Data are presented as the mean 6 SEM DaggerP 001 vscontrol P 001 vs vehicle or indicated group

Fig 7 TDT inhibited TRPC-mediated calcium influx through PKG inprimary cultured rat podocytes (A) Primary cultured podocytes from DSrats were incubated with the indicated concentrations of TDT for25 minutes cGMP concentrations were then measured and normalizedby protein amounts Data are from four independent experimentsperformed in duplicate (B and C) Cultured rat podocytes were pretreatedwith 100 nM TDT with or without TRPC inhibitor SKF96365 (10 mM) orPKG inhibitor KT5823 (1 mM) for 30 minutes followed by exposure to10 mM ATP to evoke Ca2+ influx Intracellular Ca2+ concentration wasdetermined by Fura-2 ratiometry (n = 4ndash5) (B) Ca2+ influx was measuredat the indicated time points (C) AUC of the change in calcium influx fromthe baseline was calculated Data are presented as the mean 6 SEMError bars that do not show are smaller than symbols P 005 P 001 vs vehicle group $$P 001 vs indicated group

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activator because of its potent hypotensive effect TDT couldbe a novel well-balanced renoprotective agent with respect toefficacy and safetyHowever to mention direct organ protection by TDT there

are two major limitations in this study First the measuringmethods of blood pressure tail-cuff and intra-arterial cathe-ter could mask the changes in BP since animals weresubjected to immobilization and anesthetic stress To assesschronic effect of TDT on BP in a condition closer to physiologicsettings continuous recording of BP in free-moving animalswould be needed Second it should be noted that our resultspresented here do not necessarily claim superiority of TDTover hANP because hANP was used at a single dose Tocompare the effectiveness of cardiac and renal protectionbetween TDT and hANP adequately analysis of pharmacoki-neticpharmacodynamic relationships using extra dosingamounts will be needed In addition we demonstratedthat TDT protected not only podocytes but also tubuleseven at a low dose Consistently Kato et al (2017) reportedthat systemic GC-A KO mice are more susceptible to renaldamage than podocyte-specific GC-A KO mice in a bloodpressurendashindependent manner which indicates the impor-tance of other renal cells including tubules in the signaling ofNPs Further investigations regarding the direct protection oftubules by GC-A activation will be requiredGlomerulosclerosis is caused by nuclear factor of activated

T cells (NFAT)-mediated induction of TRPC6 (Nijenhuis et al2011) As NFAT is activated by calcineurin in a calcium-dependent manner it is probable that TRPC-mediated cal-cium influx induces TRPC6 expression as a positive feedbackmechanism In this context it is important that TDT treatmentreduced TRPC activity both in vitro and in vivo contributingto the prevention of glomerulosclerosis Interestingly wealso demonstrated that TRPC-mediated calcium influx wasinhibited by PKG inhibitor consistent with the previous reportthat the renal dysfunction byGC-A gene ablation is attenuatedby a TRPC inhibitor (Staffel et al 2017)In conclusion we demonstrated that TDT exhibited cardi-

orenal protection in DS rats The generation of NEP-resistantNP using peptide engineering technology could be a promisingstrategy against cardiorenal diseases

Acknowledgments

The authors thank Shin Nippon Biomedical Laboratories LtdDrug Safety Research Laboratories (Kagoshima Japan) for theirexpertly conducted experiments

Authorship Contributions

Participated in research design Oishi Nagayama FujioConducted experiments Oishi Suzuki HasuiPerformed data analysis Oishi HommaWrote or contributed to the writing of the manuscript Oishi Obana

Fujio

References

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Arai K Morikawa Y Ubukata N Tsuruoka H and Homma T (2016) CS-3150 a novelnonsteroidal mineralocorticoid receptor antagonist shows preventive and thera-peutic effects on renal injury in deoxycorticosterone acetatesalt-induced hyper-tensive rats J Pharmacol Exp Ther 358548ndash557

Campbell DJ (2017) Long-term neprilysin inhibition - implications for ARNIs NatRev Cardiol 14171ndash186

Chen CA Hwang JC Guh JY Tsai JC and Chen HC (2006) TGF-beta1 and integrinsynergistically facilitate the differentiation of rat podocytes by increasing alpha-smooth muscle actin expression Transl Res 148134ndash141

Hashimoto Y Nakao K Hama N Imura H Mori S Yamaguchi M Yasuhara Mand Hori R (1994) Clearance mechanisms of atrial and brain natriuretic peptides inrats Pharm Res 1160ndash64

Kato Y Mori K Kasahara M Osaki K Ishii A Mori KP Toda N Ohno S KuwabaraT Tokudome T et al (2017) Natriuretic peptide receptor guanylyl cyclase-Apathway counteracts glomerular injury evoked by aldosterone through p38mitogen-activated protein kinase inhibition Sci Rep 746624

Kishimoto I Tokudome T Horio T Garbers DL Nakao K and Kangawa K (2009)Natriuretic peptide signaling via guanylyl cyclase (GC)-A an endogenous pro-tective mechanism of the heart Curr Cardiol Rev 545ndash51

Klotz S Hay I Zhang G Maurer M Wang J and Burkhoff D (2006) Development ofheart failure in chronic hypertensive Dahl rats focus on heart failure with pre-served ejection fraction Hypertension 47901ndash911

Kumagai S Nakayama H Fujimoto M Honda H Serada S Ishibashi-Ueda H KasaiA Obana M Sakata Y Sawa Y et al (2016) Myeloid cell-derived LRG attenuatesadverse cardiac remodelling after myocardial infarction Cardiovasc Res 109272ndash282

McMurray JJ Packer M Desai AS Gong J Lefkowitz MP Rizkala AR Rouleau JLShi VC Solomon SD Swedberg K et al PARADIGM-HF Investigators andCommittees (2014) Angiotensin-neprilysin inhibition versus enalapril in heartfailure N Engl J Med 371993ndash1004

Meems LMG and Burnett JC Jr (2016) Innovative therapeutics designer natriureticpeptides JACC Basic Transl Sci 1557ndash567

Moumlller CC Wei C Altintas MM Li J Greka A Ohse T Pippin JW Rastaldi MPWawersik S Schiavi S et al (2007) Induction of TRPC6 channel in acquired formsof proteinuric kidney disease J Am Soc Nephrol 1829ndash36

Nijenhuis T Sloan AJ Hoenderop JG Flesche J van Goor H Kistler AD Bakker MBindels RJ de Boer RA Moumlller CC et al (2011) Angiotensin II contributes topodocyte injury by increasing TRPC6 expression via an NFAT-mediated positivefeedback signaling pathway Am J Pathol 1791719ndash1732

Raff GL and Glantz SA (1981) Volume loading slows left ventricular isovolumic re-laxation rate Evidence of load-dependent relaxation in the intact dog heartCirc Res 48813ndash824

Schefold JC Filippatos G Hasenfuss G Anker SD and von Haehling S (2016) Heartfailure and kidney dysfunction epidemiology mechanisms and managementNat Rev Nephrol 12610ndash623

Seidelmann SB Vardeny O Claggett B Yu B Shah AM Ballantyne CM Selvin EMacRae CA Boerwinkle E and Solomon SD (2017) An NPPB promoter poly-morphism associated with elevated N-terminal pro-B-type natriuretic peptide andlower blood pressure hypertension and mortality J Am Heart Assoc 6

Solomon SD Zile M Pieske B Voors A Shah A Kraigher-Krainer E Shi VBransford T Takeuchi M Gong J et al Prospective comparison of ARNI with ARBon Management Of heart failUre with preserved ejectioN fracTion (PARAMOUNT)Investigators (2012) The angiotensin receptor neprilysin inhibitor LCZ696 in heartfailure with preserved ejection fraction a phase 2 double-blind randomised con-trolled trial Lancet 3801387ndash1395

Sonneveld R Ferregrave S Hoenderop JG Dijkman HB Berden JH Bindels RJ WetzelsJF van der Vlag J and Nijenhuis T (2013) Vitamin D down-regulates TRPC6expression in podocyte injury and proteinuric glomerular disease Am J Pathol1821196ndash1204

Staffel J Valletta D Federlein A Ehm K Volkmann R Fuumlchsl AM Witzgall R KuhnM and Schweda F (2017) Natriuretic peptide receptor guanylyl cyclase-A inpodocytes is renoprotective but dispensable for physiologic renal function J AmSoc Nephrol 28260ndash277

Takeda Y Sakata Y Higashimori M Mano T Nishio M Ohtani T Hori MMasuyama T Kaneko M and Yamamoto K (2009) Noninvasive assessment of walldistensibility with the evaluation of diastolic epicardial movement J Card Fail 1568ndash77

Volpe M (2014) Natriuretic peptides and cardio-renal disease Int J Cardiol 176630ndash639

Winn MP Conlon PJ Lynn KL Farrington MK Creazzo T Hawkins AF DaskalakisN Kwan SY Ebersviller S Burchette JL et al (2005) A mutation in the TRPC6cation channel causes familial focal segmental glomerulosclerosis Science 3081801ndash1804

Address correspondence to Dr Yasushi Fujio Laboratory of ClinicalScience and Biomedicine Graduate School of Pharmaceutical Sciences OsakaUniversity Osaka Japan 1-6 Yamadaoka Suita Osaka 565-0871 JapanE-mail fujiophsosaka-uacjp Or Shohei Oishi End-Organ Disease Labo-ratories Daiichi Sankyo Co Ltd 1-2-58 Hiromachi Shinagawa-ku Tokyo 140-8710 Japan E-mail oishishoheiprdaiichisankyocojp

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