Intradialytic complement activation precedes the development of cardiovascular events in hemodialysis patients

ORIGINAL RESEARCH published: 13 Sep embe 2018 doi: 10.3389/ immu.2018.02070 F on ie s in Immunology | www. on ie sin.o g 1Sep embe 2018 | Volume 9 | A icle 2070 Edi ed by: Nicole Thielens, UMR5075 Ins i u de Biologie S uc u ale (IBS), F ance Re iewed by: Con ad An hony Fa a , King’s College London, Uni ed Kingdom Ch is ina Ka a za e i, Uni e si y o S Ma k S John, Uni ed Kingdom *Co espondence: Felix Poppelaa s [email p o ec ed] †These au ho s ha e con ibu ed equally o his wo k Special y sec ion: This a icle was submi ed o Molecula Inna e Immuni y, a sec ion o he jou nal F on ie s in Immunology Recei ed: 04 June 2018 Accep ed: 21 Augus 2018 Published: 13 Sep embe 2018 Ci a ion: Poppelaa s F, Gaya da Cos a M, Fa ia B, Be ge SP, Assa S, Daha MR, Medina Pes ana JO, an Son WJ, F anssen CFM and Seelen MA (2018) In adialy ic Complemen Ac i a ion P ecedes he De elopmen o Ca dio ascula E en s in Hemodialysis Pa ien s. F on . Immunol. 9:2070. doi: 10.3389/ immu.2018.02070 In adialy ic Complemen Ac i a ion P ecedes he De elopmen o Ca dio ascula E en s in Hemodialysis Pa ien s Felix Poppelaa s1*†, Ma iana Gaya da Cos a1†, Be na do Fa ia1,2,3, S e an P. Be ge 1, Solmaz Assa4, Mohamed R. Daha1,5, José Osma Medina Pes ana6, Willem J. an Son1, Caspe F. M. F anssen1and Ma c A. Seelen1 1Di ision o Neph ology, Depa men o In e nal Medicine, Uni e si y o G oningen, Uni e si y Medical Cen e G oningen, G oningen, Ne he lands, 2Neph ology and In ecciology G oup, INEB/I3S, Uni e si y o Po o, Po o, Po ugal, 3Depa men o Neph ology, Hospi al B aga, B aga, Po ugal, 4Depa men o Ca diology, Uni e si y o G oningen, Uni e si y Medical Cen e G oningen, G oningen, Ne he lands, 5Depa men o Neph ology, Uni e si y o Leiden, Leiden Uni e si y Medical Cen e , Leiden, Ne he lands, 6Neph ology Di ision, Fede al Uni e si y o São Paulo, São Paulo, B azil Backg ound: Hemodialysis (HD) is a li e-sa ing ea men o pa ien s wi h end s age enal disease. Howe e , HD pa ien s ha e ma kedly inc eased a es o ca dio ascula mo bidi y and mo ali y. P e iously, a link be ween he complemen sys em and ca dio ascula e en s (CV-e en s) has been epo ed. In HD, sys emic complemen ac i a ion occu s due o blood- o-memb ane in e ac ion. We hypo hesize ha HD-induced complemen ac i a ion oge he wi h in lamma ion and h ombosis a e in ol ed in he de elopmen o CV-e en s in hese pa ien s. Me hods: HD pa ien s we e ollowed o he occu ence o CV-e en s du ing a maximum ollow-up o 45 mon hs. Plasma samples we e collec ed om 55 pa ien s a di e en ime poin s du ing one HD session p io o ollow-up. Plasma le els o mannose-binding lec in, p ope din and C3d/C3 a ios we e assessed by ELISA. In addi ion, le els o on Willeb and ac o , TNF-αand IL-6/IL-10 a ios we e de e mined. An ex- i o model o HD was used o assess he e ec o complemen inhibi ion. Resul s: Du ing median ollow-up o 32 mon hs, 17 pa icipan s de eloped CV-e en s. In he CV-e en g oup, he C3d/C3- a io sha ply inc eased 30 min a e he s a o he HD session, while in he e en - ee g oup he a io did no inc ease. In acco dance, HD pa ien s ha de eloped a CV-e en also had a sus ained highe IL-6/IL-10- a io du ing he i s 60 min o he HD session, ollowed by a g ea e ise in TNF-αle els and on Willeb and ac o a he end o he session. In he ex- i o HD model, we ound ha complemen ac i a ion con ibu ed o he induc ion o TNF-αle els, IL-6/IL-10- a io and le els o on Willeb and ac o . Conclusions: In conclusion, hese indings sugges ha ea ly in adialy ic complemen ac i a ion p edominan ly occu ed in HD pa ien s who de elop a CV-e en du ing ollow-up. In addi ion, in hese pa ien s complemen ac i a ion was accompanied Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s by a p o-in lamma o y and p o- h ombo ic esponse. Expe imen al complemen inhibi ion e ealed ha his eac ion is seconda y o complemen ac i a ion. The e o e, ou da a sugges s ha HD-induced complemen , in lamma ion and coagula ion a e in ol ed in he inc eased CV isk o HD pa ien s. Keywo ds: complemen , kidney, ca dio ascula isk, hemodialysis, biocompa ibili y, inna e immuni y, C1-inhibi o INTRODUCTION Renal eplacemen he apy (RRT) ep esen s a co ne s one in he ea men o pa ien s wi h end s age enal disease (ESRD). Hemodialysis (HD) emains he mos common o m o RRT (1). Despi e being li esa ing, HD comes wi h a isk (2). The li e expec ancy and quali y o li e o pa ien s on dialysis is in e io o he gene al popula ion. O e all, HD has been associa ed wi h inc eased ca dio ascula mo bidi y and mo ali y (3). P e ious s udies ha e sugges ed ha he inna e immune sys em plays a key ole in he de elopmen o ca dio ascula disease in HD pa ien s (4). The complemen sys em is a majo componen o inna e immuni y and ac i a ion o his sys em induces an in lamma o y esponse (5). Complemen ac i a ion can occu ia h ee pa hways: he classical pa hway (CP), lec in pa hway (LP), and al e na i e pa hway (AP). Rega dless o he igge , all pa hways lead o he clea age o C3 esul ing in he o ma ion o C3b, he la ge agmen and C3a, an anaphyla oxin. Ul ima ely, C3b is b oken down p og essi ely o iC3b and hen o he mo e s able agmen C3d. The unc ions o he complemen sys em we e hough o be limi ed o opsoniza ion and lysis o pa hogens. Howe e , nowadays his sys em is known o ha e nume ous unc ions and complemen has been shown o be in ol ed in he pa hogenesis o a ious diseases (6). Fo decades, HD has been known o ac i a e he complemen sys em (7). In dialysis, complemen ac i a ion is mainly caused by he in e ac ion o blood wi h he HD memb ane (4). Rega dless o he e o s o imp o e biocompa ibili y, complemen ac i a ion s ill occu s in HD, e en wi h mode n memb anes (8–10). I has been hypo hesized ha complemen ac i a ion leads o HD- induced in lamma ion and he eby inc eases he subsequen ca dio ascula isk (4). In acco dance, se e al s udies ha e shown an associa ion be ween complemen and ca dio ascula e en s (CV-e en ) (8,11–14). Howe e , he link be ween complemen ac i a ion p oduc s and CV-e en s emains poo ly cha ac e ized (15). Only Lines e al. epo ed an associa ion in HD pa ien s be ween soluble C5b-9 and ca dio ascula isk (15). Fu he mo e, p e ious expe imen al s udies p oposed a link be ween HD-induced complemen ac i a ion, p o-in lamma o y cy okines, and he coagula ion sys em (10,16). Abb e ia ions: AP, Al e na i e pa hway; BSA, Body su ace a ea; C5b-9, Memb ane a ack complex; CV-e en , Ca dio ascula e en ; ESRD, End s age enal disease; ELISA, Enzyme-linked immunoso ben assay; HD, Hemodialysis; IL-6, In e leukin 6; IL-10, In e leukin 10; LP, Lec in pa hway; MBL, Mannose- binding lec in; RRT, Renal eplacemen he apy; PCI, Pe cu aneous co ona y in e en ion; TNF-α, Tumo nec osis ac o -α; WF, Von Willeb and ac o We hypo hesize ha an un a o able complemen p o ile is seen in HD pa ien s who will de elop a CV-e en . To in es iga e he mechanism o inc eased ca dio ascula isk in HD, we measu ed complemen ac i a ion, p o-in lamma o y cy okines and p o- h ombo ic ac o s du ing one HD session in pa ien s ha de eloped a CV-e en du ing ollow-up and compa ed his o pa ien s wi hou a CV-e en du ing ollow-up. Fu he mo e, we used an ex- i o model o HD o u he elucida e he ole o complemen ac i a ion as a igge o in lamma ion and coagula ion in HD. MATERIALS AND METHODS S udy Popula ion and Design A coho o 55 hemodialysis pa ien s om Dialysis Cen e G oningen and he Uni e si y Medical Cen e G oningen we e ollowed o a maximum o 45 mon hs. The o iginal coho was composed ou o 109 pa ien s; howe e , due o a lack o samples only 55 pa ien s could be included o his s udy. The p o ocol has been p e iously desc ibed (2). In sho , pa ien s we e included i he du a ion o HD he apy was longe han 3 mon hs. Pa ien s wi h se e e hea ailu e (NYHA class IV) we e excluded. Pa ien cha ac e is ics we e ex ac ed om pa ien eco ds. Dialysis Se ings Pa ien s we e on main enance HD ea men o h ee imes a week wi h a low- lux polysul one hollow- ibe dialyze (F8; F esenius Ca e, Bad Hombu g, Ge many). The hemodialysis sessions las ed o 4 h. The blood and dialysa e low a es we e 250–350 and 500 mL/min, espec i ely. A cons an ul a il a ion a e was used. Dialysa e composi ion was as ollows: ace a e, 3.0 mmol/L; bica bona e, 34 mmol/L; calcium, 1.5 mmol/L; chlo ide, 108 mmol/L; glucose, 1.0 g/L; magnesium, 0.5 mmol/L; po assium, 1.0 o 2.0 mmol/L; sodium, 139 mmol/L The dialysa e empe a u e was kep on 36.0 o 36.5◦C. Blood samples we e aken jus be o e he s a o he dialysis session, and a e 30, 60, 180, and 240 min. De ini ion o Endpoin The end-poin o he s udy was de ined as he ime o he i s CV- e en . CV-e en s included ca diac, ce eb o ascula , o pe iphe al ascula e en s. Occu ence o a ca diac e en was de ined as a ischemic hea disease (uns able angina pec o is, myoca dial in a c ion, Co ona y A e y Bypass G a ing (CABG) and/o Pe cu aneous Co ona y In e en ion (PCI), sudden ca diac dea h and conges i e hea ailu e. In o de o classi y as acu e myoca dial in a c ion, wo ou o he ollowing h ee c i e ia had o be p esen : clinical s a us, ele a ed hea enzymes, and EKG changes. Ce eb o ascula e en s we e de ined as s oke, F on ie s in Immunology | www. on ie sin.o g 2Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s ischemic insul , o newly diagnosed >70% s enosis o he ex ac anial ca o id a e y. S okes and ischemic insul s had o be e i ied by CT o MRI. Pe iphe al ascula disease was de ined as ha ing in e mi en claudica ion wi h angiog aphically o sonog aphically p o en s enosis >50% o he majo a e ies o he lowe limbs o ulce s caused by a he oscle o ic s enosis o su ge y o his diso de . T ansplan a ion was a censo ing e en and he ansplan a ion da e was conside ed as he inal ollow-up da e (17). Ex- i o Model o Hemodialysis An ex- i o model o HD was used as p e iously desc ibed (18). In b ie , a closed ci cui was assembled using a pedia ic polysul one hollow- ibe dialyze (FX paed; F esenius Ca e, Ge many) and blood lines (SN-Se ONLINEplus BVM 5008-R, F esenius Ca e, Ge many). The o al olume o he ci cui was app oxima ely 50 mL. Pe usion was achie ed using a Mas e lex R pe is al ic pump (Cole-Pa me , USA) and was low-con olled (TS410 ubing low module, T ansonic sys ems Inc, USA) o each a pe usion low o app oxima ely 140 o 160 mL/min. The empe a u e was kep cons an a 37◦C and con olled by an ex e nal hea e . Whole blood was aken om heal hy olun ee s (n=3) and an icoagula ed wi h low-molecula weigh hepa in (1 U/mL). Ini ially, he ci cui was p imed wi h NaCl 0.9% and pe used o app oxima ely 20 min o emo e ai bubbles. P io o pe usion, he dialysa e compa men was illed wi h NaCl 0.9% and closed. Nex , eshly d awn hepa inized blood was added o he ci cui , while he same olume o he saline solu ion was disca ded. The sys em was pe used wi h eci cula ing blood o 4 h. Samples we e collec ed a he s a o he pe usion and a e 30, 60, 120, 180, and 240 min. To in es iga e he e ec o complemen inhibi ion, wo consecu i e sessions we e pe o med o each heal hy olun ee . Du ing one session, 200 uni s o C1-inhibi o (Cin yze©, Vi opha ma, USA) we e added o he blood p io o pe usion, whe eas he o he session wi hou C1-inhibi o (C1-INH) se ed as a con ol. In lamma o y Ma ke s and P o- h ombo ic Fac o s In he HD coho , TNF-αwas measu ed by Quan ikine HS Human Immunoassay (R&D Sys em Inc., USA). Fu hemo e, IL-6 and IL-10 we e de e mined using a quan i a i e sandwich enzyme immunoassay echnique (R&D Sys em Inc., USA). Las ly, Von Willeb and Fac o ( WF) was measu ed by enzyme- linked immunoso ben assay (Dakopa s, UK). In he ex- i o HD model, TNF-α, IL-6, IL-10, and WF we e measu ed using a human magne ic luminex assay (R&D Sys ems Inc, USA) acco ding o he manu ac u e ’s ins uc ions. Quan i ica ion o Complemen P o eins C3d was measu ed by sandwich enzyme immunoassay as p e iously desc ibed (19). Quan i a i e an igenic assay o C3 was pe o med by he adial immunodi usion echnique wi h monospeci ic an i-se a (19). The C3d/C3 a io was de e mined by di iding he C3d alues in µg/mL by he C3 concen a ion in mg/mL. Addi ionally, P ope din and MBL concen a ions we e measu ed as desc ibed ea lie (19,20). S a is ics S a is ical analysis was pe o med using IBM SPSS 22.0 (IBM Co po a ion, Chicago, IL, USA). No mally dis ibu ed da a a e p esen ed as mean ±s anda d de ia ion, whe eas non- no mally dis ibu ed da a a e shown as median wi h in e qua ile ange. Nominal da a a e displayed as o al numbe o pa ien s wi h pe cen age [n(%)]. Di e ences be ween wo g oups we e assessed wi h he s uden - es , whe eas he pai ed - es was used o compa e alues o a single a iable du ing di e en ime poin s wi hin he HD session. A one-way ANOVA was used when assessing o di e ences in mul iple g oups, ollowed by Bon e oni’s pos -hoc compa isons es s. The associa ion be ween di e en a iables and he incidence o CV-e en we e assessed by Cox p opo ional haza d eg ession. The Ha ell’s C s a is ic is he equi alen o he a ea unde he ROC cu e, i he ou come is bina y (21). E hics This s udy was pe o med in acco dance o he Decla a ion o Helsinki and was app o ed by he Medical E hical Commi ee om he Uni e si y Medical Cen e G oningen. All pa icipan s signed in o med w i en consen . RESULTS Pa ien s Cha ac e is ics Blood samples om 55 pa ien s on main enance HD we e a ailable, o which 35 we e male and 20 emale (Table 1). The mean age was 62 ±15 yea s and baseline dialysis in age was 1.2 yea s [IQR: 0.6–3.9 yea s]. The median ollow-up o he s udy was 32 mon hs and du ing his ime 17 pa ien s (31%) de eloped a CV-e en , whe eas 16 pa ien s died (29%). In ou s udy, he causes o dea h we e ca dio ascula (44%), in ec ion (12.5%), discon inua ion o he HD ea men (12.5%), o unknown (31%). Among he pa ien s ha de eloped CV-e en s, 35% had acu e co ona y synd ome, 17% needed co ona y a e y bypass su ge y, 11% de eloped conges i e hea ailu e, 17% had a ce eb o- ascula acciden and 17% de eloped pe iphe al ascula disease. Nex , we c ea ed wo di e en g oups; he 17 pa ien s ha de eloped a CV-e en du ing ollow-up (CV-e en g oup) and he 38 pa ien s ha emained e en - ee (e en - ee g oup). Complemen Ac i a ion in he HD Pa ien s To assess complemen ac i a ion, we de e mined he C3d/C3- a io in 55 pa ien s du ing one HD session a he s a o he ollow-up. The C3d/C3- a io a baseline was no s a is ically di e en be ween he pa ien s ha would de elop a CV-e en (7.0 ±6.2) compa ed o he pa ien s ha would no (9.0 ± 7.4). Su p isingly, a he end o he HD session he C3d/C3- a io was also no s a is ically di e en be ween he wo g oups (CV- e en g oup: 11.8 ±8.5, e en - ee g oup: 12.9 ±10.0). Howe e , when he in adialy ic C3d/C3- a ios we e compa ed be ween he wo g oups, clea di e ences we e seen (Figu e 1 and Table 1). A 30 min in adialysis, he e was a signi ican inc ease in he C3d/C3- a io in he CV-e en g oup compa ed o he pa ien s who emained e en - ee. Du ing hese ini ial 30 min, he C3d/C3- a io inc eased by 3.29 old in he CV-e en g oup and F on ie s in Immunology | www. on ie sin.o g 3Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s TABLE 1 | Baseline cha ac e is ics o ou s udy popula ion o hemodialysis pa ien s wi h and wi hou a ca dio ascula e en . Pa ien s Uni a iable All (n=55) CV-e en (n=17) No CV-e en (n=38) P* S . be a P# C3d/C3 RATIO 0 min 5.9 [4.8–9.3] 5.6 [4.7–8.1] 6.1 [4.8–11.5] 0.2 −0.02 0.6 30 min 9.8 [7.3–17.0] 12.0 [10–33.5] 8.4 [6.8–13.4] 0.01 0.05 0.002 60 min 8.4 [5.4–12.4] 7.8 [4.4–11.9] 8.9 [5.6–16.2] 0.2 −0.04 0.2 120 min 8.6 [5.5–14.5] 7.4 [5.2–12.7] 9.1 [5.6–16.4] 0.3 −0.03 0.3 240 min 8.4 [5.8–17.9] 8.6 [5.9–20.0] 8.3 [5.8–17.9] 0.8 −0.04 0.8 DEMOGRAPHICS Age, yea s 62.7 ±15.8 66.5 ±11.4 61.0 ±17.2 0.2 0.01 0.3 Male gende , n (%) 35 (63) 11 (64) 24 (63) 0.9 −0.22 0.6 Cu en diabe es, n (%) 10 (18) 5 (29) 5 (13) 0.2 0.51 0.3 Hype ension, n (%) 48 (87) 14 (82) 34 (89) 0.2 −0.99 0.1 Ca dio ascula his o y, n (%) 11 (20) 5 (29) 6 (15) 0.2 0.64 0.2 BSA, m21.8 ±0.3 1.7 ±0.5 1.9 ±0.2 0.08 −2.04 0.02 HEMODIALYSIS Dialysis in age, mon hs 14.1 [7.3–47.3] 12.2 [8.6–50.4] 16.4 [4.7–47.5] 0.5 0.002 0.8 Ul a il a ion olume, L 2.5 ±0.7 2.4 ±0.6 2.5 ±0.7 0.9 0.00 0.7 Ul a il a ion a e, ml/kg/h 8.1 ±2.3 7.9 ±1.8 8.2 ±2.5 0 7 −0.01 0.8 PRIMARY RENAL DISEASE, n(%) Hype ension 12 (22) 4 (23) 8 (21) 0.8 0.02 0.9 Diabe es 5 (9) 3 (17) 2 (5) 0.1 0.65 0.3 ADPKD 7 (13) 1 (6) 6 (16) 0.3 −0.96 0.3 FSGS 6 (11) 2 (12) 4 (10) 0.8 0.89 0.2 IgA neph opa hy 3 (6) 0 (0) 3 (7) 0.2 −3.15 0.4 Ch onic pyeloneph i is 1 (2) 1 (6) 0 (0) 0.1 2.02 0.06 Glome uloneph y is 6 (11) 1 (6) 5 (13) 0.4 −0.75 0.4 O he Diagnosis 9 (16) 3 (18) 6 (16) 0.8 0.35 0.6 Unknown 5 (9) 2 (12) 3 (7) 0.8 −0.34 0.9 LABORATORY MEASUREMENTS Hema oc i , % 34.4 ±3.7 34.0 ±4.2 34.6 ±3.5 0.6 −5.98 0.3 HbA1C, mmol/mol 5.56 ±0.9 5.7 ±1.1 5.5 ±0.8 0.4 0.17 0.4 Albumin, g/L 38 [37-41] 38 [37-41] 38 [36-42] 0.9 0.004 0.9 Calcium, mmol/L 2.32 ±0.2 2.31 ±0.1 2.32 ±0.2 0.8 −0.55 0.6 Phospha e, mmol/L 1.74 ±0.5 1.70 ±0.4 1.75 ±0.6 0.7 0.009 0.9 hsCRP, mg/L 5.5 [1.5–8.8] 5.0 [2.3–11.8] 5.8 [1.4–8.8] 0.5 0.01 0.3 MEDICATION Aspi in, n (%) 34 (62) 8 (47) 26 (68) 0.2 −0.80 0.09 Calcium channel blocke s, n (%) 12 (22) 4 (23) 8 (21) 0.8 −0.14 0.7 β-Blocke , n (%) 31 (56) 10 (59) 21 (55) 0.8 0.24 0.6 ACE inhibi o , n (%) 5 (9) 1 (5) 4 (10) 0.6 −0.44 0.6 AT2– ecep o an agonis s, n (%) 8 (14) 2 (12) 6 (16) 0.7 −0.41 0.6 S a in, n (%) 13 (24) 2 (12) 11 (29) 0.2 −0.88 0.2 Diu e ics, n (%) 5 (9) 2 (12) 3 (7) 0.7 1.11 0.2 P*indica es P- alue o he di e ence in baseline cha ac e is ics be ween he pa ien wi h and wi hou a ca dio ascula -e en . Di e ences we e es ed by S uden ’s -Tes o Mann– Whi ney U es o con inuous a iables and wi h χ2 es o ca ego ical a iables. Da a a e p esen ed as mean ±SD o median [IQR]. P#indica es P- alue o uni a ia e Cox- eg ession o he occu ence o CV-e en . Da a a e p esen ed as be a coe icien wi h co esponding P- alue. CV, ca dio ascula ; BSA, body su ace a ea; ADPKPD, au osomal dominan polycys ic disease; FSGS, ocal segmen al glome uloscle osis; HbA1c, Hemoglobin A1c; hsCRP, high sensi i e C- eac i e p o ein; ACE inhibi o , angio ensin-con e ing-enzyme inhibi o ; AT2- ecep o an agonis s, Angio ensin II ecep o an agonis s. by only 1.26 old in he e en - ee g oup (P<0.01). In addi ion, Cox eg ession analysis was pe o med o assess he associa ion be ween C3d/C3 a io a 30 min and occu ence o a CV-e en (Table 2). In he c ude model, C3d/C3 a ios we e associa ed wi h a haza d a io o 1.06 (95% CI 1.02-1.09; P<0.001). A e adjus men o age and gende , a iables wi h P<0.1 F on ie s in Immunology | www. on ie sin.o g 4Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s FIGURE 1 | The C3d/C3- a io du ing hemodialysis. Cou se o plasma C3d/C3 a io in pa ien s ha de eloped a ca dio ascula e en (CV-e en ) du ing ollow-up and in hose ha emained CV-e en ee (no CV-e en ). The da a is p esen ed as mean ±SEM and C3d/C3- a io was calcula ed by di iding he C3d alues (µg/mL) by he C3 le els (in mg/mL). The C3d/C3- a io was de e mined a he s a o hemodialysis session and 30, 60, 180 and 240 min a e . Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e C3d/C3 a ios a di e en ime poin s wi hin one g oup (**P<0.01). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. The numbe o subjec is 17 in he “CV-e en g oup” and 38 in he “No CV-e en g oup”. TABLE 2 | Associa ions o in adialy ic complemen ac i a ion wi h ou come. Ca dio ascula e en s C3d/C3 a io a 30 min HR 95% CI P Model 1 1.06 1.02–1.09 <0.001 Model 2 1.06 1.03–1.09 0.001 Model 3 1.04 1.01–1.08 0.03 Model 4 1.06 1.02–1.09 0.001 Model 5 1.07 1.02–1.09 0.002 Model 1: c ude. Model 2: adjus ed o age and gende . Model 3: adjus ed o BSA, aspi in and p ima y ch onic pyeloneph i is. Model 4: adjus ed o DM, ca dio ascula his o y and hype ension. Model 5: adjus ed o HD in age , UF a e and UF olume. Da a a e p esen ed as haza d a io (HR) plus 95% con idence in e al (CI). BSA, body su ace a ea; DM, diabe es melli us; HD, Hemodialysis; UF, ul a il a ion a e. in uni a ia e analysis (BSA, ch onic pyeloneph i is as p ima y enal disease and use o aspi in), ca dio ascula isk ac o s (CV his o y, DM and hype ension) o cha ac e is ics o HD (Ul a il a ion a e, ul a il a ion olume and dialysis in age), he associa ion be ween C3d/C3 a io a 30 min and CV-e en emained signi ican . Subsequen ly, he Ha ell’s-C s a is ics was de e mined o u he con i m he po en ial ela ionship be ween complemen ac i a ion and CV-e en s. Plasma C3d/C3 a io a 30 min had a Ha ell’s-C s a is ics o 0.71 (95% CI 0.55–0.88; P=0.01). We nex se ou o assess he con ibu ion o he AP and LP o HD-induced complemen ac i a ion. Due o a lack o samples, p ope din and MBL le els we e measu ed in a subg oup o 30 pa ien s (Figu e 2). In his subg oup, he e we e 11 pa ien s in he CV-e en g oup and 19 pa ien s in he e en ee g oup. MBL and p ope din le els we e compa able be ween he wo g oups a he s a and a he end o he HD session. Con e sely, a 30 min in adialysis, MBL le els dec eased signi ican ly in he e en - ee g oup bu no in he CV-e en g oup (P<0.05). Fu he mo e, p ope din le els we e signi ican ly lowe a 30 min in he CV- e en g oup, compa ed o he e en - ee g oup. To summa ize, MBL consump ion was seen in he e en - ee g oup implying LP ac i a ion, while lowe p ope din le els we e obse ed in he CV-e en g oup sugges ing AP ac i a ion. In lamma o y and P o- h ombo ic Fac o s in he HD Pa ien s We de e mined cy okines and Von Willeb and ac o ( WF) o in es iga e i complemen ac i a ion du ing HD was accompanied by a p o-in lamma o y esponse and a p o- h ombo ic s a e. Du ing HD, dis inc ime-cou ses o le els o WF we e obse ed be ween he wo g oups (Figu e 3). In he CV-e en g oup, WF le els inc eased s eadily du ing he session F on ie s in Immunology | www. on ie sin.o g 5Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s FIGURE 2 | In adialy ic le els o p ope din and Mannose-binding lec in. Cou se o plasma mannose-binding lec in (MBL) and p ope din in pa ien s ha de eloped a ca dio ascula e en (CV-e en ) du ing ollow-up and in hose ha emained CV-e en ee (no CV-e en ). The da a is p esen ed as mean ±SEM. (A) The le els o MBL we e de e mined a he s a o hemodialysis session and 30 and 240 min a e . (B) The le els o p ope din we e de e mined a he s a o hemodialysis session and 30 and 240 min a e . Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e le els a di e en ime poin s wi hin one g oup (*P<0.05). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. The numbe o subjec is 11 in he “CV-e en g oup” and 19 in he “No CV-e en g oup”. (P<0.05). Fu he mo e, compa ed o he e en - ee g oup, he CV-e en g oup had signi ican ly highe le els o WF a 180 and 240 min in adialysis (P<0.05). Cy okines such as umo nec osis ac o -α(TNF-α) may ini ia e in lamma ion and a e he e o e belie ed o play a ole in dialysis- ela ed ca dio ascula isk. Le els o TNF-α ose signi ican ly du ing he HD session in bo h g oups (Figu e 4A). In he CV-e en g oup, le els peaked a 180 min a e he s a o he HD session (P<0.01) and we e signi ican ly highe han in he e en - ee g oup (P<0.05). Fu he mo e, in he e en - ee g oup, he maximum TNF-α le els we e eached a he end o he session (P<0.001). To e alua e he ela ion be ween an i-in lamma o y cy okines and p o-in lamma o y cy okines, we de e mined he IL-6/IL- 10 a io (Figu e 4B). In e es ingly, IL-6/IL-10 a ios we e he highes in bo h g oups a he s a o he HD session and showed a dec easing end du ing he dialysis session, al hough no signi ican compa ed o baseline. Mo eo e , a 60 min in adialysis an impo an dec ease in he IL-6/IL-10 a io occu ed in he e en - ee g oup, indica ing a shi owa d a less in lamma o y p o ile. Howe e , IL-6/IL-10 a ios emained ele a ed in he HD pa ien s ha de eloped a CV-e en du ing ollow-up, e ealing a signi ican di e ence be ween he g oups a his ime poin (P<0.05). O e all, enhanced le els o p o- in lamma o y and p o- h ombo ic media o s seem o p elude he de elopmen o CV-e en s in HD pa ien s. Ex- i o Model o Hemodialysis To u he e alua e he e ec o HD-induced complemen ac i a ion on in lamma ion and coagula ion, we used an ex- i o model o HD. Du ing he 4 h o pe usion, he C3d/C3 a io inc eased p og essi ely om 4.7 ±0.6 a baseline o 55.8 ± 12.5 a e 240 min (Figu e 5A). MBL and p ope din le els we e de e mined o disc imina e be ween complemen ac i a ion ia he AP and/o he LP. Bo h MBL and p ope din le els dec eased FIGURE 3 | Le els o on Willeb and ac o du ing hemodialysis. Cou se o on Willeb and ac o ( WF) in pa ien s ha de eloped a ca dio ascula e en (CV-e en ) du ing ollow-up and in hose ha emained CV-e en ee (no CV-e en ). The da a is p esen ed as mean ±SEM. WF was de e mined a he s a o hemodialysis session and 60, 180 and 240 min a e he s a o he session. Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e le els a di e en ime poin s wi hin one g oup (*P<0.05, **P<0.01). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. The numbe o subjec is 17 in he “CV-e en g oup” and 38 in he “No CV-e en g oup”. signi ican ly o e ime. A e 4 h, MBL le els we e educed by 55.2% (P<0.05) and p ope din le els by 34.4%, espec i ely (Figu e 5). We nex assessed in lamma o y and p o- h ombo ic ac o s. Simila ly o complemen ac i a ion, he HD model esul ed in a signi ican inc ease in TNF-α, IL-6/IL-10 a io, and WF le els a e 240 min o dialysis (Figu e 6). F on ie s in Immunology | www. on ie sin.o g 6Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s Finally, we e alua ed he e ec o complemen inhibi ion in ou model o es i complemen ac i a ion ac s as a igge o in lamma ion and coagula ion in HD. C1-INH was added and signi ican ly educed C3d/C3 a ios compa ed o con ols, namely om 55.8 ±13 o 33.1 ±24 (Figu e 5A;P<0.05). HD- induced consump ion o p ope din and MBL was no p e en ed by he use o C1-INH.Fu he mo e, TNF-αle els we e 1654 ±631 ng/mL a e 240 min in he con ol session, while in he session wi h C1-INH le els we e educed o 48.7 ±74.7 ng/mL(Figu e 6A). Co espondingly, a simila end was seen a e 240 min in he IL-6/IL-10 a io (con ol session 1086 ±630 pg/mL, C1-INH session 51 ±78 pg/mL; Figu e 6C) and o WF le els (con ol session: 98.2 ±22.7 pg/mL, C1-INH session: 1.7 ±3.3 pg/mL) when he con ol session was compa ed o C1-INH session (Figu e 6B). To summa ize, C1-INH addi ion was able o inhibi HD-induced complemen ac i a ion and he eby educe WF, TNF-αand IL-6/IL-10 by 98, 97, and 95% espec i ely. DISCUSSION Hemodialysis ea men comes wi h he balance be ween he dange s o ad anced u emia and he inhe en isks ela ed o his o m o RTT (22,23). The highe ca dio ascula isk seen in his popula ion is no only ela ed o ESRD bu i is also associa ed wi h he HD p ocedu e i sel (2). Inna e immuni y has been p oposed o be he missing link in he mechanism o CV-e en s in HD pa ien s (4). We obse ed dis inc di e ences in molecula p o iles du ing HD o pa ien s ha will la e de elop CV-e en s compa ed o hose who emained e en - ee du ing ollow-up. A he s a o dialysis, a unique peak in complemen ac i a ion was only seen in pa ien s in he CV-e en g oup. Fu he mo e, enhanced in lamma ion and coagula ion accompanied he complemen ac i a ion seen in HD pa ien ha will de elop CV-e en s. Mo eo e , hese p ocesses a ose long be o e he ac ual de elopmen o he CV-e en . Al oge he hese h ee elemen s showed di e en dynamics, wi h complemen ac i a ion possibly ini ia ing hese p ocesses. In acco dance, complemen inhibi ion in ou ex- i o model no only dec eased complemen ac i a ion bu also diminished p o-in lamma o y and p o- h ombo ic media o s. Despi e signi ican ad ances in he biocompa ibili y o HD memb anes, complemen ac i a ion emains an undesi ed bu ele an issue (8,15). Highe le els o complemen componen s as well as loss o complemen inhibi o s ha e been associa ed wi h a highe isk o ca dio ascula disease in HD pa ien s (8,11– 14). Recen ly, complemen ac i a ion p io o a HD session was associa ed wi h he occu ence o CV-e en s in HD pa ien s (15). He e, we showed ha ac i a ion o C3 du ing dialysis is linked o he de elopmen o CV-e en s. Ou s udy is he i s , o ou knowledge, o assess he ela ionship be ween in adialy ic complemen ac i a ion and subsequen ou come. In acco dance, p e ious s udies ha e shown ha ac i a ion o he complemen sys em peaks du ing he i s 15 o 30 min o he HD session (24). Howe e , he mechanism by which complemen ac i a ion inc eases he isk o ca dio ascula disease emains la gely unknown. The LP and AP ini ia e complemen ac i a ion du ing HD (25,26). In ou s udy, we only ound MBL consump ion in he e en - ee g oup, implying ha his dec ease is ac ually bene icial. In acco dance, MBL has been p oposed o be in ol ed in he emo al o a he ogenic pa icles, he eby dec easing a he oscle osis. Ou p e ious da a showed ha highe MBL le els in HD pa ien s we e associa ed wi h p o ec ion agains ca dio ascula disease (9). We also ound a ise in p ope din le els in he e en - ee g oup. P ope din, unlike o he complemen ac o s, is p oduced by leukocy es, p edomina ely neu ophils (27). The e o e, he inc ease in p ope din is p esumably he esul o leukocy e ac i a ion by he HD memb ane leading o deg anula ion (28). Since, his ise was no seen in he CV-e en g oup, we specula e his was due o p ope din consump ion by AP ac i a ion in hese pa ien s. We ound highe TNF-αle els and IL-6/IL-10 a ios in pa ien s ha would de elop a CV-e en . TNF-αand IL-6 a e po en cy okines ha can ini ia e a powe ul p o-in lamma o y eac ion (29,30). I his esponse is no con ained, i can lead o hypo ension, o gan dys unc ion, and e en ually esul in dea h. Ele a ed le els o hese cy okines ha e also been ela ed o an inc eased isk o CV-e en s in he gene al popula ion and in HD pa ien (31–34). In con as , IL-10 is a majo an i-in lamma o y cy okine wi h he abili y o supp ess he p oduc ion and sec e ion o p o-in lamma o y media o s in leukocy es, he eby e ec i ely con olling he in lamma ion (35). The IL-6/IL-10 a io has p e iously been linked o ou come a e in lamma o y diso de s and o he de elopmen o HD- induced le en icula dys unc ion (36–38). In ex- i o models, he induc ion o IL-6 du ing he bio-incompa ibili y eac ion was shown o be comple ely complemen -dependen , while he induc ion o TNF-αwas only pa ially complemen -dependen (16). In addi ion, in a p ima e model o HD, complemen inhibi ion lead o enhanced le els o IL-10, demons a ing he ela ionship be ween he wo sys ems (39). Th ombosis is a key elemen in he de elopmen o ca dio ascula disease. P e iously, Péqué iaux e al. epo ed ha WF is a good p edic o o CV-e en s in pa ien s unde going RRT (40). Von Willeb and ac o is a glycop o ein in ol ed in hemos asis bu WF is also a ma ke o endo helial cell ac i a ion (41). We ound signi ican ly highe le els o WF in he g oup o pa ien s who de eloped CV-e en s, which could be e idence o a p o h ombo ic s a e. WF is p oduced in endo helial cells and megaka yocy es, bu also s o ed in he g anules o pla ele s (42). Conside ing ou ex- i o lacks endo helial cells, he WF is mos likely de i ed om pla ele s. The elease o WF could ei he be he di ec e ec o complemen ac i a ion o ia C5a-ac i a ed leukocy es (43,44). The link be ween he complemen sys em and h ombosis is no new in HD (45). Complemen ecep o s on leukocy es a e impo an o he o ma ion o pla ele - leukocy es complexes, which con ibu es o h ombo ic p ocesses (46). In addi ion, complemen ac i a ion du ing HD induces he p oduc ion o p o-coagula ion ac o s (47). Mo eo e , plasma le els o C3 co ela ed wi h a dense clo s uc u e in HD pa ien s (48). The complemen sys em is a s ong media o o he bioincompa ibili y eac ion. The e o e, we p oposed ha F on ie s in Immunology | www. on ie sin.o g 7Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s FIGURE 4 | Le els o umo nec ose ac o alpha and he a io o in e leukin-6 o in e leukin-10 du ing hemodialysis. Cou se o umo nec ose ac o alpha (TNF-α) and a io o in e leukin-6 (IL-6) o in e leukin-10 (IL-10) in pa ien s ha de eloped a ca dio ascula e en (CV-e en ) du ing ollow-up and in hose ha emained CV-e en ee (no CV-e en ). The da a is p esen ed as mean ±SEM. (A) The le els o TNF-αwe e de e mined a he s a o hemodialysis session and 60, 180 and 240 min a e he s a o he session. (B) Le els IL-6 and IL-10 we e de e mined a he s a o hemodialysis session and 60, 180, and 240 min a e . The IL-6/IL- 10 a io was calcula ed by di iding he IL-6 (in pg/mL) alues by he IL-10 le els (in pg/mL). Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e le els a di e en ime poin s wi hin one g oup. (*P<0.05, **P<0.01, ***P<0.001). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. The numbe o subjec is 17 in he “CV-e en g oup” and 38 in he “No CV-e en g oup”. FIGURE 5 | Complemen le els du ing ex i o hemodialysis. Two di e en sessions we e pe o med wi h whole blood o h ee heal hy dono s; one session wi h C1-inhibi o (C1-INH) and one session wi hou , he con ol session. The da a is p esen ed as mean ±SEM. (A) C3d/C3 a ios we e measu ed o de e mine complemen ac i a ion. (B) MBL le els signi ican ly dec ease o e ime du ing he session. (C) P ope din le els we e educed du ing he session, al hough no signi ican ly. Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e le els a di e en ime poin s wi hin one g oup (*P<0.05, **P<0.01). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. F on ie s in Immunology | www. on ie sin.o g 8Sep embe 2018 | Volume 9 | A icle 2070 Poppelaa s e al. Complemen Ac i a ion in HD Linked o CV-E en s FIGURE 6 | Le els o umo nec ose ac o alpha, a io o in e leukin-6 o in e leukin-10 and WF du ing ex i o hemodialysis. Two di e en sessions we e pe o med wi h whole blood o h ee heal hy dono s; one session wi h C1-inhibi o (C1-INH) and one session wi hou , he con ol session. The da a is p esen ed as mean ± SEM. (A) TNF-αle els signi ican ly inc eased in he con ol session, bu no in he session wi h C1-INH. (B) Co espondingly, WF showed a signi ican ise in he con ol session, while C1 INH addi ionlead o signi ican ly lowe le els. (C) The IL-6/IL-10 a io p og essi ely inc eased o e ime, al hough no s a is ically signi ican . Di e ences be ween he wo g oups we e assessed by he s uden - es and a one-way ANOVA ollowed by Bon e oni’s pos -hoc compa isons es s was used o compa e le els a di e en ime poin s wi hin one g oup (*P<0.05). The hash ag abo e he ba s deno es a signi ican di e ence be ween he wo g oups (#P<0.05), whe eas he as e isk abo e he ba s deno es a signi ican di e ence compa ed o baseline wi hin he g oup. complemen ac i a ion has an essen ial ole in o ches a ing he in lamma o y esponse in HD. In acco dance wi h he esul obse ed in he HD pa ien s; ou ex- i o model demons a ed ha he dialyze induces complemen ac i a ion, in lamma ion and enhances coagula ion. We hen wonde ed i HD-induced in lamma ion could be a enua ed by complemen inhibi ion. Addi ion o C1-INH o he ex- i o HD model signi ican ly diminished HD-induced complemen ac i a ion and also almos comple ely abolished he induc ion o TNF-αle els, L-6/IL-10 a ios and WF le els. Simila ly, Kou zelis e al, also demons a ed in an ex i o model o HD he induc ion o coagula ion du ing 2 h o pe usion. In hei s udy, comps a in was used o block complemen ac i a ion a he le el o C3 (10). We pos ula e ha HD-induced complemen ac i a ion esul s in he o ma ion o anaphyla oxins, he eby esul ing in he ac i a ion o pe iphe al blood mononuclea cell and pla ele s ini ia ing a p o-in lamma o y and p o- h ombo ic esponse. P e iously, se e al epo s demons a ed ha bioincompa ibili y-induced in lamma ion elies mainly on complemen , whe eas g anulocy e enzyme elease was p edominan ly C3-dependen , leukocy e ac i a ion and p o- h ombo ic media o s we e la gely dependen on C5 (16,46). Al oge he , ou esul s suppo he hypo hesis o he complemen sys em as a key componen in HD-induced in lamma ion and coagula ion, which subsequen ly leads o a highe isk o CV-e en s. We a e awa e ha ou s udy has s eng hs and limi a ions. Al hough he s udy has a long ollow up, he samples we e only collec ed du ing a single hemodialysis session. Ou s udy could ha e bene i ed om a second assessmen o hese pa ame e s du ing ano he dialysis session in he same pa ien s, o assess ep oducibili y and inc ease eliabili y. Fu he mo e, while complemen ac i a ion was seen in he pa ien s du ing HD as well as in ou ex- i o model, clea di e ences we e p esen . Mo eo e , in pa ien s a signi ican peak o complemen ac i a ion was seen du ing he i s 30 min o HD. In con as , in ou ex- i o model a con inuous ise o complemen ac i a ion was seen un il he end o he session. Ob iously hese disc epancies a ise due o he di e ences o in- i o o ex- i o. Fo ins ance, in he ex- i o model blood eci cula es wi hou e-en e ing he human body, he e o e i lacks he in e ac ion wi h endo helial cells, li e and o he o gans. Las ly, he size o ou coho could be conside ed small and he e o e migh impac he s a is ical analysis. Howe e , due o he long ollow up, we achie ed a ela i ely high numbe o CV-e en s which inc eases he powe o he s udy in he compa isons be ween he CV-e en g oup and he e en ee g oup. The e is a g owing body o da a suppo ing a ole o he complemen sys em in he de elopmen o ca dio ascula disease. Ekdahl e al. p oposed ha complemen ac i a ion ini ia es an in lamma o y cascade and ampli ies p o- h ombo ic p ocesses (4). Fo he i s ime, o ou knowledge, we demons a ed in adialy ic di e ences in complemen ac i a ion, in lamma ion and a p o- h ombo ic ac o in HD pa ien s ha will de elop a CV-e en compa ed o HD pa ien s ha will no . Fu he mo e, we showed ha complemen inhibi ion du ing HD esul ed in dec eased le els o he p o-in lamma o y and p o- h ombo ic media o s. Fu u e s udies ha e o de e mine wha he ideal a ge is o inhibi complemen in HD o a enua e hese p ocesses and o de e mine i his dec eases he isk o CV-e en s in HD pa ien s. AUTHOR CONTRIBUTIONS FP, MG, MD, CF, and MS esea ch idea and s udy design. FP, MG and SA da a acquisi ion. FP, MG, BF, SB, MD, JM, W S, CF, and MS da a analysis/in e p e a ion. FP and MG s a is ical analysis and w o e he manusc ip . All au ho s we e in ol ed in edi ing he inal manusc ip . All au ho s ead and app o ed he inal manusc ip . F on ie s in Immunology | www. on ie sin.o g 9Sep embe 2018 | Volume 9 | A icle 2070