Endothelin A receptor blocker and calcimimetic in the adenine rat model of chronic renal insufficiency

RESEARCH ARTICLE Open Access Endo helin A ecep o blocke and calcimime ic in he adenine a model o ch onic enal insu iciency Su i Tö mänen 1 , Ilkka Pö s i 1,2,8* , Päi i Lakkis o 3,4 , Ilkka Tikkanen 3,5 , Onni Niemelä 1,6 , Timo Paa onen 1,7 , Jukka Mus onen 1,2 and A u E ä an a 1 Abs ac Backg ound: We s udied whe he endo helin ecep o an agonis and calcimime ic ea men s in luence enal damage and kidney enin-angio ensin (RA) componen s in adenine-induced ch onic enal insu iciency (CRI). Me hods: Male Wis a a s (n= 80) we e di ided in o 5 g oups o 12 weeks: con ol (n= 12), 0.3% adenine (Ade; n= 20), Ade + 50 mg/kg/day si axen an (n= 16), Ade + 20 mg/kg/day cinacalce (n = 16), and Ade + si axen an + cinacalce (n = 16). Blood p essu e (BP) was measu ed using ail-cu , kidney his ology was examined, and RA componen s measu ed using RT-qPCR. Resul s: Adenine caused ubuloin e s i ial damage wi h se e e CRI, anemia, hype phospha emia, 1.8- old inc ease in u ina y calcium exc e ion, and 3.5- old and 18- old inc eases in plasma c ea inine and PTH, espec i ely. Si axen an alle ia ed ubula a ophy, while si axen an + cinacalce combina ion educed in e s i ial in lamma ion, ubula dila a ion and a ophy in adenine- a s. Adenine die did no in luence kidney angio ensin con e ing enzyme (ACE) and AT 4 ecep o mRNA, bu educed mRNA o enin, AT 1a ,AT 2 , (p o) enin ecep o and Mas o 40–60%, and supp essed ACE2 o 6% o ha in con ols. Si axen an educed BP by 8 mmHg, c ea inine, u ea, and phospha e concen a ions by 16–24%, and PTH by 42%. Cinacalce did no in luence BP o c ea inine, bu educed PTH by 84%, and inc eased hemoglobin by 28% in adenine- a s. The ea men s u he educed enin mRNA by 40%, while combined ea men no malized plasma PTH, u ina y calcium, and inc eased ACE2 mRNA 2.5- old e sus he Ade g oup (p<0.001). Conclusions: In adenine-induced in e s i ial neph i is, si axen an imp o ed enal unc ion and ubula a ophy. Si axen an and cinacalce educed kidney enin mRNA by 40%, while hei combina ion alle ia ed ubuloin e s i ial damage and u ina y calcium loss, and inc eased kidney issue ACE2 mRNA. Keywo ds: Ch onic kidney disease, Si axen an, Cinacalce , C ea inine, Pa a hy oid ho mone, Renal enin-angio ensin sys em Backg ound Ch onic kidney disease (CKD) and i s p og ession o end s age enal disease (ESRD) emain a global clinical chal- lenge [1, 2]. Rega dless o he o iginal kidney insul , one o he majo causes leading o he decline o enal unc ion is in e s i ial ib osis [3, 4]. Adenine die adminis a ion o a s, and he subsequen deposi ion o 2,8-dihyd oxyade- nine c ys als in kidney issue, induce an in lamma o y e- sponse esembling he pa hology o in e s i ial neph i is and he consequen ib osis [5–7]. The adenine model has been shown o esul in se e e ch onic enal insu iciency (CRI) wi h i s ypical u emic indings such as ele a ed c e- a inine and u ea, anemia, and seconda y hype pa a hy - oidism (SHPT) [5, 8–11]. Ac i a ion o he enin-angio ensin sys em (RAS) is, by a , he bes -cha ac e ized p omo e o in lamma ion and ib osis in he pa hology o CRI [12, 13]. The o ma ion * Co espondence: [email p o ec ed] 1 Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland 2 Depa men o In e nal Medicine, Tampe e Uni e si y Hospi al, Tampe e, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Tö mänen e al. BMC Neph ology (2017) 18:323 DOI 10.1186/s12882-017-0742-z o local angio ensin II is known o p omo e in lamma ion and ib osis [12, 13], while inhibi ion o RAS a enua es p o einu ia, glome uloscle osis and also he de elopmen o in e s i ial ib osis [14]. Howe e , he need o addi ional enop o ec i e he apeu ics is e iden , and hus he endo helin sys em has ecen ly ga ne ed high in e es . The endo helin sys em is ac i a ed in i ually all causes o CKD, in which endo helin ecep o A (ETA) ac i a ion p omo es asocons ic ion, enal cell inju y, in lamma ion, and ib osis. The endo helin sys em is also linked o RAS by posi i e eedback loops [15–17]. While ETA an agonis s ha e been shown o amelio a e enal inju y, ib osis, p o- einu ia, and disease p og ession in expe imen al diabe ic, hype ensi e, and emnan kidney a models o CKD [16], less is known abou he e ec s o ETA an agonism in enal diseases o ubuloin e s i ial o igin. Some s udies sugges ha ETA an agonism migh de e io a e enal unc ion in polycys- ic models o CKD [18]. Dis u bed calcium-phospha e me abolism and SHPT may also con ibu e o he p og ession o CKD [19]. We p e iously ound ha die a y phospha e loading, and phospha e binding by o al calcium ca bona e ea men , al e ed he con en s o RAS componen s in he kidney and ao a, and also in luenced glome uloscle osis and ubuloin e s i ial damage in 5/6 neph ec omized a s [20–22]. Howe e , he key playe modula ing RAS com- ponen s h ough changes in calcium-phospha e me abol- ism has emained elusi e. Recen ly, educing se um pa a hy oid ho mone (PTH) wi h cinacalce , a posi i e allos e ic modula o o he calcium sensing ecep o (CaSR), in adenine-induced a model o CKD was epo ed o a enua e enal ib ogenesis and educe plasma c ea in- ine concen a ion [10]. I is plausible ha some o he e - ec s o o al calcium ca bona e supplemen a ion and he posi i e allos e ic modula ion o CaSR, i.e. calcimime ics, migh be media ed ia local CaSR ac i a ion. Recen ly, majo in e es has been di ec ed owa ds he possible bene i s o endo helin ype A ecep o blockade in kidney diseases wi h glome ula damage and p o einu ia [16]. He e we e alua ed he e ec s o ea men wi h he selec i e ETA an agonis si axen- an and he posi i e allos e ic CaSR agonis cinacalce , alone and in combina ion, on he p og ession o ad- enine a model o se e e in e s i ial neph i is. As bo h endo helin sys em and calcium-phospha e me abolism can a ec in a enal RAS, we hypo he- sized ha he pu a i e posi i e ou comes o si axen an and cinacalce migh be e lec ed as changes in he kidney componen s o RAS, ans o ming g ow h ac o -ß1 (TGF-ß1) and connec i e issue g ow h ac- o (CTGF), key ma ke s and po en ial he apeu ic a ge s o kidney ib osis [23]. To ou knowledge, he local kidney issue RAS componen s ha e no been p e iously s udied in his model o CKD. Me hods Animals and expe imen al design Eigh y male Wis a a s (Ha lan Labo a o ies, Ho s , The Ne he lands) aged 8 weeks we e housed ou o a cage wi h ee access o wa e and chow. A he age o 10 weeks he a s we e di ided in o 5 g oups wi h ma ched sys olic blood p essu es (BP) and body weigh s. The sys olic BP was mea- su ed a +28°C by he ail-cu me hod (Model 129 Blood P essu e Me e ; IITC Inc., Woodland Hills, CA). One g oup was gi en he con ol die (RM3, Scanbu , Ka lslunde, Denma k) and 4 g oups ecei ed 0.3% aden- ine (Sigma-Ald ich, Sain Louis, MO) added o he chow. We chose he con inuous 0.3% adenine die o his 12-week s udy [11], based on (1) p e ious indings o high mo ali y ela ed o he 0.75% adenine die , e en i adenine was wi hd awn a e 4 weeks o adminis a- ion [24]; and (2) conce ns abou he e e sibili y o CRI when adenine adminis a ion is discon inued [9, 24]. In addi ion o adenine, a s in 3 g oups ecei ed ei he si axen an (50 mg/kg/d added o he d inking wa e p o- ec ed om ligh ; P ize , New Yo k, NY), cinacalce (20 mg/kg/d added o he 0.3% adenine chow; Amgen Thousand Oaks, CA), o bo h o hese ea men s o 12 weeks. The doses o hese compounds we e chosen on he basis o p e ious epo s [10, 25–29]. The s udy g oups we e: con ol (Con ol, n= 12), adenine (Ade, n= 20), adenine + si axen an (Ade + S, n= 16), aden- ine + cinacalce (Ade + C, n = 16), and adenine + com- bina ion ea men (Ade + SC, n = 16). The medica ions we e con inued un il he end o he s udy. Body weigh s we e moni o ed weekly, sys olic BP was measu ed a he end o he s udy, and 24-h wa e con- sump ion, u ine ou pu , and chow consump ion we e moni o ed wice du ing he ea men s (weeks 5 and 10) in me abolic cages. U ine samples we e s o ed a −70°C o analyses. A close o he s udy, a s we e anes he ized wi h in ape i oneal u e hane (1.3 g/kg), he ca o id a e y was cannula ed, and blood samples we e d awn wi h EDTA and hepa in as an icoagulan s, as app op ia e. Plasma and weighed issue samples we e s o ed a −70 °C. The expe imen al design was app o ed by he Animal Expe imen a ion Commi ee o he Uni e si y o Tampe e, and he P o incial Go e nmen o Wes e n Finland, Depa men o Social A ai s and Heal h, Finland; iden i ica ion numbe ESLH-2008-03943/Ym- 23. The in es iga ion con o ms o he Guiding P inciples o Resea ch In ol ing Animals. Sample size calcula ion As he majo aim o he s udy was o e alua e i he ea - men s can amelio a e adenine-induced CRI, c ea inine was chosen as he p ima y ou come a iable. The ele an di e ence in he means o c ea inine alues o ea ed and un ea ed adenine a s as well as he s anda d de ia ion o Tö mänen e al. BMC Neph ology (2017) 18:323 Page 2 o 13 c ea inine alues we e bo h es ima ed a 40 μmol/l. The es ima ion o s anda d de ia ion was de i ed om p e i- ously published adenine a expe imen s [5]. Powe o he s udywasse a 0.80and he ypeIe o p obabili ya 0.05. The sample size o un ea ed adenine a s was adjus ed o expec ed a i ion o 20% and, hus, he sample size was mul iplied by 1.25 in he Ade g oup. The acqui ed sample sizes we e 15 and 19 o ea ed and un ea ed ad- enine a s, espec i ely, and we e ul ima ely ounded up o he nea es e en numbe ( a s we e housed ou o a cage). The sample size was calcula ed using PS p og am [30]. Ho monal and chemical analyses Sodium, po assium, c ea inine, u ea, phospha e, calcium, choles e ols and p o eins we e measu ed using s anda d clinical chemical me hods (Cobas In eg a 800 Clinical Chemical Analyze , Roche Diagnos ics, Basel, Swi ze land). The de e mina ion o 24-h c ea inine clea ance was based on u ine de e mina ions om week 10 and plasma samples aken a close o he s udy. The enzyma ic me hod used he e o c ea inine de e mina ion has been p e iously shown o be eliable in bo h heal hy and diseased a s [31]. Hemoglobin was de e mined pho ome ically (Technicon H*2™, Technicon Ins u- men s Co po a ion, Ta y own, NY) and a in ac pa a hy oid ho mone (PTH) le els by immuno adio- me ic assay (Immu opics Inc. San Clemen e, CA). Renal his ology Mo phological in es iga ions om haema oxylin-eosin s ained kidney sec ions we e pe o med wi h ligh mi- c oscopy in a blinded manne . Glome ula and ubuloin- e s i al sco es we e de e mined acco ding o Schwa z e al. [32], o iginally desc ibed by El Nahas e al. [33] and Venian e al. [34], espec i ely. Semi-quan i a i e glome ula sco e o each g oup was assessed by examining 100 glome uli a magni ica ions ×100 and ×400: g ade 0, no mal glome uli; g ade 1, mesangial expansion; g ade 2, mild/mode a e segmen al hyalinosis/scle osis in ol ing <50% o he glome ula u ; g ade 3, di use glome ula hyalinosis/scle osis in ol ing ≥50% o he u ; g ade 4, di use glome uloscle osis wi h o al u obli e a ion and collapse. The index in each g oup was exp essed as he mean o all sco es ob ained. The ubuloin e s i ial sco es ( ubula a ophy, ubula dila a ion, in e s i ial expansion, in e s i ial in lamma ion and in e s i ial ib osis) we e de e mined by isualizing andomly selec ed en ields pe kidney a a magni ica- ion ×100. The g ading was as ollows: g ade 0, no changes; g ade 1, lesions in ol ing <25% o he a ea; g ade 2, lesions in ol ing 25–50%; g ade 3, lesions a - ec ing >50% o he a ea; and g ade 4, lesions in ol ing (almos ) he en i e a ea. The means o all sco es gi en comp ised he ubuloin e s i ial indexes. Real- ime quan i a i e RT-PCR To al RNA was isola ed om kidney issue using T izol eagen (In i ogen, Ca lsbad, CA). Re e se ansc ip ion o RNA was pe o med using M-MLV e e se ansc ip - ase (In i ogen) acco ding o he manu ac u e ’s ins uc- ions. Be a-ac in was used as a housekeeping gene. PCRs we e pe o med wi h SYBR G een o TaqMan chemis y using ABI PRISM 7000 sequence de ec ion (Applied Biosys ems, Fos e Ci y, CA). PCRs o angio ensin con e ing enzyme (ACE), angio ensin II ecep o ype 1 a (AT 1aR ), angio ensin IV ecep o (AT 4R ), (p o) enin ecep o (PRR), and TGF-ß1 we e pe o med in duplica e in 25 μl inal olume con aining 1X SYBR G een Mas e mix (Applied Biosys ems) and 300 nM o p ime s. PCRs o ACE2, angio ensin II ecep o ype 2 (AT 2R ), enin, and CTGF we e pe o med in duplica e in 25 μl inal olume con ain- ing 1X TaqMan Mas e mix (Applied Biosys ems), 300 nM o p ime s ( o enin 900 nM) and 100 nM o ACE2, 150 nM o AT 2R , 250 nM o enin, o 200 nM o CTGF TaqMan p obe, espec i ely. PCR cycling condi ions o mRNAs we e 10 min a +95 °C and 40 cycles o 20 s a +95 °C and 1 min a +60 °C. Da a we e analyzed using he absolu e s anda d cu e me hod [35]. Be a-ac in (TaqMan assay code Rn00667869_m1) was analyzed in a simila ashion o 18S analysis in ou ea lie pape [20]. The exp essions o be a-ac in did no di e be ween he g oups, and he o he esul s we e no malized o he le els o be a-ac in. Wes e n blo ing o TGF-ß1 Kidney issues we e homogenized in a lysis bu e con ain- ing 100 mmol/L NaCl, 10 mmol/L KCl, 8 mmol/L Na 2 HPO 4 ,3mmol/LMgCl 2 , 0.5% NP40, 10 mmol/L T is- HCl, pH 7.4 and p o ease inhibi o s (Comple eTM Mini, P o ease Inhibi o Cock ail Table s, Roche Diagnos ics GmbH, Mannheim, Ge many). Homogena es we e incu- ba ed on ice o 30 min and cen i uged a 15000 g o 15 min. P o ein concen a ion in he supe na an was de e mined using he BCA p o ein assay ki (Pie ce, Rock o d, IL, USA). Equal amoun s o p o ein (25 μg) we e ac iona ed by 4–20% Mini-PROTEAN TGX S ain- F ee Gel (Bio-Rad, He cules, CA, USA). The gel was s ain- ee ac i a ed and blo ed on o a low- luo escence poly inylidene luo ide memb ane (LF PVDF T ans-Blo Tu bo RTA T ans e Ki ) wi h T ans-Blo Tu bo T ans e Sys em (Bio-Rad). The memb ane was hen s ain- ee imaged o o al p o ein no maliza ion using ChemiDoc Touch Imaging Sys em (Bio-Rad) and p obed wi h mouse an i-TGF-ß1 (R&D Sys ems, Minneapolis, MN, USA). Enhanced chemiluminescen de ec ion was pe o med using he Supe Signal Wes Pico Chemiluminescen Subs a e (The mo Scien i ic, Rock o d, IL, USA) and he chemiluminescen applica ion on he ChemiDoc Tö mänen e al. BMC Neph ology (2017) 18:323 Page 3 o 13 Touch Imaging Sys em. Quan i ica ion o signal in en- si ies was pe o med using he ImageLab so wa e (Bio-Rad) by no malizing he in ensi ies o speci ic bands o he o al p o ein con en on he memb ane. Rep esen a i e o iginal Wes e n blo image o TGF-ß1 is shown in Addi ional ile 1: Figu e S1. Da a p esen a ion and analysis o esul s S a is ical analysis was ca ied ou using one-way ana- lysis o a iance (ANOVA), and pos -hoc compa isons we e pe o med wi h he Tukey HSD es i he a iables had equal a iances e i ied by he Le ene’s es . I his c i e ion was no me , he K uskal-Wallis es and he Mann-Whi ney es wi h he Bon e oni co ec ion we e used. The esul s in he able we e p esen ed as mean ± SEM, while he igu es show mean ± 95% con i- dence in e al o he mean. Di e ences we e conside ed signi ican when P< 0.05. SPSS 17.0 so wa e (SPSS Inc., Chicago, IL, USA) was used o he s a is ics. Resul s Animal da a The adenine die esul ed in dec eased body weigh s and lowe chow in ake, bu no a s we e los du ing he s udy (Table 1). Chow in ake in he Ade + S g oup was educed sligh ly less han in he Ade g oup. The adenine die did no ele a e sys olic BP, whe eas bo h si axen an- ea ed g oups showed educed BP when compa ed wi h he Ade g oup. The olumes o d inking luid and u ine we e inc eased in all adenine g oups, while he Ade + SC g oup showed lowes u ine olumeamong headenine- ea edg oups. Labo a o y indings All adenine-die g oups showed ma kedly ele a ed plasma c ea inine and u ea concen a ions (Fig. 1a-b). In he Ade g oup plasma c ea inine and u ea we e 3.5 and 4.5- old highe , espec i ely, han in he Con ol g oup. Si axen an ea men alone and in combina ion wi h cinacalce ame- lio a ed he inc ease in c ea inine and u ea when com- pa ed wi h he Ade g oup. C ea inine clea ance in he Ade g oup was educed o abou 22%, and in he si axen an- ea ed g oups o abou 32%, o ha in he Con ol g oup (Table 1). Cinacalce ea men alone did no in luence plasma c ea inine o u ea concen a ions e sus he Ade g oup (Fig. 1a and b). Hemoglobin was ma kedly educed in he Ade g oup when compa ed wi h he Con ol g oup (Fig. 1c). Si axen an ea men did no a ec hemoglobin, while cinacalce ea - men alone and in combina ion wi h si axen an ele a ed hemoglobin by 28% when compa ed wi h he Ade g oup. The e we e no di e ences in 24-h u ine p o ein exc e ion among he s udy g oups (Fig. 1d). Plasma sodium and po assium concen a ions we e in- c eased in all adenine g oups, while plasma sodium le el Table 1 Animal da a and labo a o y indings in he s udy g oups Con ol Ade Ade + S Ade + C Ade + SC Numbe o animals 12 20 16 16 16 Body weigh (g) week 0 406 ± 8 409 ± 5 409 ± 5 409 ± 5 409 ± 5 week 12 450 ± 11 339 ± 6* 341 ± 6* 340 ± 7* 359 ± 8* Chow in ake a week 10 (g/24 h) 20.3 ± 1.0 13.9 ± 0.3* 16.5 ± 0.5*†15.1 ± 0.7* 14.8 ± 0.8* Sys olic blood p essu e (mmHg) week 0 139 ± 1 138 ± 1 139 ± 1 138 ± 1 139 ± 1 week 9 137 ± 2 138 ± 1 130 ± 2†138 ± 1‡129 ± 2*† D inking olume (ml/24 h) 31.0 ± 1.9 87.5 ± 2.9* 88.1 ± 1.8* 83.5 ± 3.5* 81.2 ± 2.4* U ine olume (ml/24 h) 20.0 ± 2.3 73.9 ± 2.2* 82.7 ± 2.7*‡69.1 ± 3.8*‡54.7 ± 2.5*† 24-h c ea inine clea ance (μl/min/kg) a 379 ± 23 82 ± 6* 119 ± 8*†96 ± 11* 122 ± 13*† Final plasma Sodium (mmol/l) 135.1 ± 0.7 138.3 ± 0.5* 141.5 ± 0.6*†140.1 ± 0.5* 140.7 ± 0.5*† Po assium (mmol/l) 3.63 ± 0.09 4.34 ± 0.07* 4.22 ± 0.08* 4.19 ± 0.12* 4.05 ± 0.07* Choles e ol (mmol/l) 1.45 ± 0.05 2.32 ± 0.10* 1.78 ± 0.09†1.99 ± 0.10*‡1.61 ± 0.05† HDL (mmol/l) 0.60 ± 0.02 0.91 ± 0.04* 0.70 ± 0.02†0.82 ± 0.04* 0.69 ± 0.02*† T igly (mmol/l) 1.06 ± 0.14 1.14 ± 0.10 0.86 ± 0.10 0.98 ± 0.08‡0.68 ± 0.04† Non-HDL (mmol/l) 0.86 ± 0.04 1.41 ± 0.07* 1.08 ± 0.08†1.17 ± 0.07*‡0.92 ± 0.04† P o eins (g/l) 53.4 ± 1.0 51.9 ± 0.8 52.8 ± 1.2 51.1 ± 1.5 51.9 ± 0.9 Mean ± SEM; Ade, 0.3% adenine die ; S, si axen an 50 mg/kg/day; C, cinacalce 20 mg/kg/day; a es ima ed om u ine collec ion du ing week 10 and inal plasma c ea inine; *p< 0.05 s. Con ol; †p< 0.05 s. Ade; ‡p< 0.05 s. Ade + SC Tö mänen e al. BMC Neph ology (2017) 18:323 Page 4 o 13 was sligh ly u he ele a ed by si axen an (Table 1). Plasma o al, HDL, and non-HDL choles e ol concen a- ions we e inc eased in he Ade g oup, and we e no sig- ni ican ly in luenced by cinacalce ea men . Howe e , plasma o al and non-HDL choles e ol concen a ions did no di e om he Con ol g oup in he si axen an- ea ed g oups. Plasma p o ein concen a ions did no di e be ween any o he s udy g oups (Table 1). Plasma o al calcium concen a ion was no a ec ed by he adenine die (Fig. 2a), bu was sligh ly highe in he Ade + S han in he Ade + SC g oup. Plasma phos- pha e was signi ican ly inc eased in all adenine- ed a s (Fig. 2b), while he Ade + S a s showed lowe phospha e le els han he Ade + SC a s. Plasma PTH was in- c eased 18- old in he Ade g oup when compa ed wi h he Con ol g oup (Fig. 2c). Si axen an ea men alone educed plasma PTH concen a ion by 42% when com- pa ed wi h he Ade g oup, while plasma PTH in he wo cinacalce - ea ed g oups did no signi ican ly di e om ha in he Con ol g oup. The 24-h u ina y calcium ex- c e ion was inc eased abou 2- old in all o he adenine g oups e sus he Con ol g oup, wi h he excep ion o he combined si axen an + cinacalce g oup, in which u ina y calcium exc e ion did no di e om ha in he Con ol g oup (Fig. 2d). Renal his ology The deposi s o 2,8-dihyd oxyadenine c ys als we e accom- panied by clea ubuloin e s i ial damage in all adenine- ed a s (Figs. 3 and 4). In con as , he glome uli o he adenine g oups showed only mino al e a ions, so ha he glome u- loscle osis index was sligh ly highe in all adenine g oups combined han in he Con ol g oup (Fig. 3a). The adenine die inc eased in e s i ial in il a ion o in lamma o y cells when compa ed wi h he Con ol g oup (Figs. 3b and 4). The combina ion o si axen an and cinacalce alle ia ed in e s i ial in lamma ion when compa ed wi h un ea ed Ade a s, while he in luence o cinacalce mono he apy was no signi ican (p= 0.054 o Ade + C s. Ade). In e s i ial ib osis was mode a ely inc eased in all adenine- ed a s, and was no a ec ed by he ea men s (Figs. 3c and 4). The adenine die in- duced expansion o he in e s i ial issue ( e lec ing in- lamma ion, ib osis and edema), while cinacalce alone and in combina ion wi h si axen an alle ia ed in e s i ial issue expansion (Figs. 3d and 4). Fig. 1 Ba g aphs show plasma c ea inine (a), u ea (b), blood hemoglobin (c), and 24-h u ine p o ein exc e ion (d) in a s inges ing no mal die (Con ol; n= 12), 0.3% adenine die (Ade; n= 20), Ade + 50 mg/kg/day si axen an (Ade + S; n= 16), Ade + 20 mg/kg/day cinacalce (Ade + C; n = 16), and Ade + si axen an + cinacalce (Ade + SC; n = 16); mean ± con idence in e al; * P< 0.05 s. Con ol, † P< 0.05 s. Ade, ‡P< 0.05 s. Ade + SC Tö mänen e al. BMC Neph ology (2017) 18:323 Page 5 o 13 All adenine g oups showed inc eased sco es o ubula dila a ion and a ophy when compa ed wi h he Con ol g oup, while he combina ion o cinacalce and si axen an alle ia ed bo h ubula dila a ion and a ophy. Si axen an ea men alone also alle ia ed ubula a ophy, while he e ec o cinacalce mono he apy was no signi ican (p= 0.095) (Figs. 3e- and 4) Renal RAS, CTGF and TGF-ß1 mRNAs, and TGF-ß1 p o ein Renal enin mRNA le els we e s ongly educed by he adenine die , while he ea men s esul ed in u he 40–45% educ ion in enin mRNA when compa ed wi h he Ade g oup (Fig. 5a). PRR mRNA con en s we e e- duced in all adenine- ed g oups by abou 40–50%, bu no addi ional e ec was obse ed wi h he ea men s (Fig. 5b). Renal ACE mRNA con en was no a ec ed by adenine adminis a ion, bu was mode a ely educed in he Ade + S g oup e sus he Con ol g oup (Fig. 5c). In con as , kidney ACE2 mRNA con en was s ikingly supp essed (by 85–94%) in all adenine g oups when compa ed wi h he Con ol g oup (Fig. 5d). The sup- p ession o ACE2 mRNA, howe e , was less ma ked in he g oup ea ed wi h si axen an + cinacalce when compa ed wi h he o he adenine g oups, and ACE2 con en in he Ade + SC g oup was 2.5- old highe han in he Ade g oup (p< 0.001). Renal AT 1aR mRNA con en was dec eased by abou 50% in all adenine-die g oups when compa ed wi h he Con ol g oup (Fig. 6a). Kidney AT 2R mRNA was also educed by abou 50% in he adenine g oups (Fig. 6b), al hough signi ican di e ence was no eached be ween any single g oup e sus he Con ol g oup. AT 4R mRNA emained unchanged by adenine eeding (Fig. 6c). Mas ecep o mRNA in he adenine g oups was abou 40% lowe han ha in he Con ol g oup (Fig. 6d), while again no signi ican di e ence was obse ed be ween any single g oup e sus he Con ol g oup. Nei he si axen an no cinacalce ea men in luenced enal issue AT 1aR ,AT 2R ,AT 4R , o Mas mRNA con en s (Fig. 6a-d). CTGF mRNA con en was inc eased in he Ade g oup, while all ea men s es o ed CTGF mRNA o he le el o he Con ol a s (Fig. 7a). TGF-ß1 mRNA con en was inc eased in he Ade and Ade + S g oups, and sligh ly u he ele a ed in he wo cinacalce - ea ed Ade-g oups (Fig. 7b). When de e - mined using Wes e n blo ing, TGF-ß1 p o ein was clea ly inc eased by he adenine-die , and was no a ec ed by si axen an o cinacalce ea men s. The di e ence in Fig. 2 Ba g aphs show plasma calcium (a), phospha e (b), PTH (c), and 24-h u ine calcium exc e ion (d); s udy g oups as in Fig. 1; mean ± con idence in e al; * P< 0.05 s. Con ol, † P< 0.05 s. Ade, ‡P< 0.05 s. Ade + SC Tö mänen e al. BMC Neph ology (2017) 18:323 Page 6 o 13 kidney issue TGF-ß1 p o ein con en was no signi ican be ween he Con ol and Ade + SC g oups due o he la ge a iabili y in he TGF-ß1 signal in he la e g oup (Fig. 7c). Discussion This s udy in es iga ed he e ec s o si axen an and cina- calce , alone and in combina ion, on he p og ession o CRI and kidney RAS componen s in he adenine a model o CRI. The 0.3% adenine die o 12 weeks induced se e e CRI co esponding o s age 4 CKD, wi h ypical u emic indings including anemia, hype phospha emia, SHPT, and ele a ed plasma po assium concen a ion [36]. The a s ecei ing adenine also showed weigh loss, in- c eased wa e consump ion and polyu ia, in conce wi h p e ious indings in his model [5, 10, 11]. Howe e , sys- olic BP and u ina y p o ein exc e ion we e no inc eased. We applied he ail-cu me hod o measu e sys olic BP, and wi h his app oach we ha e p e iously ound ele a ed BP in he 5/6 neph ec omy model o CRI [20–22]. By means o adio eleme y, a mo e accu a e me hod o measu e BP, a mode a e 18 mmHg ele a ion o mean a - e ial p essu e was epo ed in adenine- ed a s [37]. How- e e , he le el o CRI in hose a s co esponded o s age 5 CKD wi h 90% educ ion in c ea inine clea ance [36], whe eas in he p esen s udy he educ ion was Fig. 3 Ba g aphs show indices o glome uloscle osis (a), in e s i ial in lamma ion (b), in e s i ial ib osis (c), in e s i ial expansion (d), ubula dila a ion (e), and ubula a ophy ( ); s udy g oups as in Fig. 1; mean ± con idence in e al; * P< 0.05 s. Con ol, † P< 0.05 s. Ade Tö mänen e al. BMC Neph ology (2017) 18:323 Page 7 o 13 app oxima ely 80%. Inc eased p o einu ia has p e iously been epo ed in a s ed 0.75% adenine die o 4 weeks, ollowed by he wi hd awal o adenine o 8 weeks, how- e e , in ha s udy he a s also had se e e s age 5 CKD wi h a 10- old inc ease in plasma c ea inine [10]. Thus, he abo e disc epancies can be pa ially explained by a ia ions in he le el o enal insu iciency due o he di e en p o ocols o adenine adminis a ion. Al oge he , he absence o p o einu ia and he p esen mo phological indings showing ubuloin e s i ial issue damage ollowing adenine adminis a ion indica e ha his model is associa ed wi h low le el o glome ula Fig. 4 Rep esen a i e pho omic og aphs o haema oxylin-eosin s ained kidney his ology in he s udy g oups; Con ol (a), Ade (b), Ade + S (c), Ade + C (d), Ade + SC (e); 1 = glome uli, 2 = heal hy issue ( ubules); 3 = adenine c ys al o ma ions; 4 = dila ed ubule; 5 = ubula a ophy; 6 = in e s i ial in lamma ion Fig. 5 Ba g aphs show enin (a), (p o) enin ecep o (b), angio ensin con e ing enzyme (c), and angio ensin con e ing enzyme 2 (d) mRNA con en s in kidney issue de e mined using eal- ime RT-qPCR; enin mRNA exp ession (a) is shown ela i e o be a-ac in exp ession; s udy g oups as in Fig. 1; mean ± con idence in e al; * P< 0.05 s. Con ol, † P< 0.05 s. Ade, ‡P< 0.05 s. Ade + SC Tö mänen e al. BMC Neph ology (2017) 18:323 Page 8 o 13 damage. This no ion is suppo ed by simila his opa ho- logical indings in p e ious a and mice adenine models o CKD [5, 38]. O no e, in he p esen s udy plasma p o- ein concen a ions we e simila in all a g oups, and his a gues agains he iew ha he adenine-die would ha e caused ma ked ex acellula luid accumula ion. The p esen s udy showed ha selec i e ETA an agonism wi h si axen an a 50 mg/kg/day mode a ely imp o ed enal unc ion in his non-p o einu ic model o ad anced in e s i- ial neph i is. The ac s ha a simila imp o emen in c e- a inine clea ance was obse ed in bo h si axen an- ea ed g oups, and ha no a s we e los du ing he s udy, s eng hen his inding. Si axen an also educed ubula a - ophy, while he combina ion o si axen an + cinacalce e- duced in e s i ial in lamma ion, in e s i ial expansion, ubula dila a ion and ubula a ophy in he adenine- ea ed a s. The inhibi ion o he di ec e ec s o he ETA ecep o is he ob ious mechanism o he educed p o- g ession o CRI and imp o ed mo phology in he si axen an- ea ed a s, as ETA ac i a ion is known o p o- mo e enal cell inju y, in lamma ion and ib osis [16, 17]. P e iously ea men wi h endo helin ecep o an agonis s has amelio a ed enal inju y, ib osis, p o einu ia, and dis- ease p og ession in expe imen al diabe ic, hype ensi e, and emnan kidney a models o CKD [16]. Ou esul s indica e ha selec i e ETA an agonism is also bene icial in his in e s i ial model o ch onic neph i is. Al hough he p esen model o CRI was no hype en- si e, si axen an adminis a ion educed BP. As BP was educed o le els lowe han in no mal con ols in spi e o he p e ailing CRI, his e ec was a he a ibu ed o he di ec e ec s o ETA an agonism han o he bene i- cial in luences on enal unc ion. Due o he asodila ing e ec s, si axen an in luences kidney hemodynamics [15–17], and he bene icial e ec s on enal unc ion may ha e pa ially been media ed ia inc eased enal blood low. The possibili y emains ha si axen an in e e ed wi h he adenine-induced enal inju y ia i s e ec s on kidney blood low and ubula unc ion, as such mechanisms may ha e subsequen ly in luenced he o ma ion o adenine c ys als in he ubula luid. Si axen an ea men also educed plasma phospha e, PTH, o al choles e ol, and non-HDL choles e ol concen a ions. The dis u bances o calcium-phospha e me abolism a e associa ed wi h he le el o impai men in kidney unc ion [36]. In addi ion, CRI is cha ac e ized by abno mali ies in he composi ion and me- abolism o plasma lipop o eins [39, 40], and a signi ican educ ion o lipop o ein ca abolism is obse ed in ad- anced enal insu iciency [40]. The e o e, he bene icial e ec s o si axen an on phospha e, PTH and lipid Fig. 6 Ba g aphs show angio ensin II ecep o ype 1 a (a), angio ensin II ecep o ype 2 (b), angio ensin II ecep o ype 4 (c), and Mas oncogene (d) mRNA con en s in kidney issue de e mined using eal- ime RT-qPCR; s udy g oups as in Fig. 1; mean ± con idence in e al; * P< 0.05 s. Con ol, † P< 0.05 s. Ade, ‡P< 0.05 s. Ade + SC Tö mänen e al. BMC Neph ology (2017) 18:323 Page 9 o 13