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