INFLAMACIÓ SISTÈMICA I CIRROSI HEPÀTICA. IMPLICACIÓ EN EL PRONÒSTIC I FISIOPATOLOGIA DE LA INSUFICIÈNCIA HEPÀTICA AGUDA SOBRE CRÒNICA I DE LA SÍNDROME HEPATORENAL Universitat de Barcelona Facultat de Medicina i Ciències de la Salut Departament de Medicina Línia de recerca: Fetge, sistema digestiu i metabolisme. Barcelona, 2019 Directors: Dr. Pere Ginès i Gibert Cap de Servei d’Hepatologia Institut de Malalties Digestives i Metabòliques Hospital Clínic de Barcelona Catedràtic del Departament de Medicina Universitat de Barcelona Dra. Elsa Solà Vergés Especialista del servei d’Hepatologia Institut de Malalties Digestives i Metabòliques Hospital Clínic de Barcelona Autora: Cristina Solé Martí Especialista en Hepatologia Institut de Malalties Digestives i Metabòliques Hospital Clínic de Barcelona
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INFLAMACIÓ SISTÈMICA I CIRROSI HEPÀTICA. IMPLICACIÓ EN EL PRONÒSTIC I FISIOPATOLOGIA DE LA INSUFICIÈNCIA
HEPÀTICA AGUDA SOBRE CRÒNICA I DE LA SÍNDROME HEPATORENAL
Universitat de Barcelona Facultat de Medicina i Ciències de la Salut
Departament de Medicina Línia de recerca: Fetge, sistema digestiu i metabolisme.
Barcelona, 2019
Directors: Dr. Pere Ginès i Gibert Cap de Servei d’Hepatologia
Institut de Malalties Digestives i Metabòliques
Hospital Clínic de Barcelona
Catedràtic del Departament de Medicina
Universitat de Barcelona
Dra. Elsa Solà Vergés Especialista del servei d’Hepatologia
Institut de Malalties Digestives i Metabòliques
Hospital Clínic de Barcelona
Autora: Cristina Solé Martí Especialista en Hepatologia
Institut de Malalties Digestives i Metabòliques
Hospital Clínic de Barcelona
Aquesta tesi no hagués estat possible sense l’ajuda, la col·laboració i la
presència de vàries persones. M’agradaria expressar la més sincera gratitud.
Pere, en primer lloc gràcies per la confiança que vas tenir en mi, perquè em vas
deixar entrar al grup i des de llavors m’has guiat pel camí de la recerca i m’has
ensenyat a ser millor metge. Admiro l’immens coneixement que tens, la facilitat
en explicar i fer fàcil conceptes complexes, i la capacitat per capgirar els
problemes o resultats i fer-ne un repte. Alhora, tens en compte el valor de la
vida personal. De doctorands n’hi ha molts, però de mentor només se’n té un i
et marca per sempre. Així que moltes gràcies.
Elsa, m’has ajudat, has col·laborat i corregit tots els meus projectes, m’has
ensenyat recerca i estadística pas a pas, m’has fet part dels teus estudis i has
valorat sempre la feina que feia. Vaig tenir l’honor de substituir-te i gaudir de la
feina que fem, però també recordo el temps que passàvem juntes a la consulta
i a la RAE abans que marxessis. Vas marxar i hem seguit en contacte ja per
feina, per Skypes o trucades més personals. Ets la meva companya de
congressos i a més, una bona amiga. Moltes gràcies per tot.
Equip Helios (Patri, Isa, Glòria, Rebeca, Marta Carol, Roser, Nicki, Elisa, Núria,
Marta Cervera, Xavi i Sonia), sense cap dubte aquesta tesi no hagués estat
possible sense vosaltres. L’esforç, el treball, la dedicació i el granet de cadascú
de vosaltres fa que les coses es puguin dur a terme i a més, que surtin bé. Feu
també, que el dia a dia sigui agradable. Patri, co-becària, vam treballar molt,
però també recordo molt bons moments, de ball, de plors i riures. Gràcies a
tots!
Als becaris italians (Laura, Alessandro i Salvatore), ha sigut fantàstic tenir-vos.
Heu treballat com un més i m’heu acompanyat en moments vitals. Grazie mille!
Als del lab; gràcies per ajudar-me, per atendre tots els dubtes i col·laborar en
els projectes. Marta, gràcies per la paciència, per fer que entengués una mica
més el món de la microbiota, de la investigació i el laboratori en general.
Als de digestiu de Bellvitge. A tots els adjunts i residents, que són companys i
amics, però en especial a l’Ari i a l’Alba, gràcies perquè vam compartir tota la
residència, inclòs l’últim any de decisions, que un d’ells era el repte d’embarcar-
se a fer la tesi. Em vau animar que l’emprengués i m’hi heu acompanyat fins
avui. Gràcies a més, per l’amistat que hem anat construint i que ara és tant
sòlida.
Lixa, Buxe, Cris, Maria, i a les de la uni (Heura, Mariona, Elba, Laura, Gemma i
Ana), perquè sou les millors amigues que algú es pot imaginar. M’heu escoltat,
m’heu preguntat i m’heu deixat explicar. Gràcies per compartir els dubtes, pors,
alegries i pel costat que sempre em feu. Gràcies.
Pare, Mare, Joan i Xavi, a vosaltres perquè sempre hi sou, perquè creieu en mi
i em feu creure que sóc capaç de tot, perquè m’heu fet sempre costat, perquè
m’aconselleu i m’escolteu, i en resum, perquè m’estimeu tant. Gràcies.
Clara, nina, tu m’has donat tota l’energia i empenta per poder acabar la tesi.
Ets el més bonic que ens ha passat mai. Així que gràcies també.
Carlos, aquesta tesi va dedicada especialment a tu, que m’has acompanyat en
tot aquest projecte personal, em vas recolzar i animar a emprendre’l, m’has
escoltat, m’has donat consells, sempre m’has encoratjat a que seguís i m’has
reforçat que la decisió que havia pres era la correcte. Sé la sort que tinc, ets la
millor parella i el millor company de vida que es pot tenir. T’estimo. Gràcies.
ÍNDEX
AJUDES A LA INVESTIGACIÓ.......................................................................... 8
LT Limfòcit T LB Limfòcit B NK Natural Killer Mf Macròfags PMN Polimorfonuclears Cels Cèl·lules MHC Complex Major d’Histocompatibilitat Ig Immunoglobulina PRRs Pattern recognition receptors DAMPs Damage-associated molecular patterns PAMPs Pathogen-associated molecular patterns PCR Proteïna C reactiva MELD Model of end-stage liver disease AKI Acute Kidney injury PBE Peritonitis bacteriana espontània ACLF Acute-on-chronic liver failure SHR Síndrome hepatorenal SHP Síndrome hepatopulmonar DILI Dany hepàtic per tòxics/ Drug incuced liver injury
11
1. INTRODUCCIÓ
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1. INTRODUCCIÓ 1.1. Generalitats La cirrosi és la fase final de les malalties cròniques del fetge i és una causa
creixent de morbiditat i mortalitat en països desenvolupats1. Es considera
l’onzena causa de mort en adults a nivell mundial, causant 1.16 milions de
morts per any al món i aproximadament 150.000 morts per any a Europa2,3.
L’etiologia de la cirrosi varia segons l’àrea geogràfica, a Europa, les principals
causes són l’alcohol, el virus de la hepatitis C i B, destacant l’increment del
fetge gras no alcohòlic (NAFLD)2,3. La cirrosi i el carcinoma hepatocel·lular són
les indicacions més freqüents de trasplantament hepàtic, realitzant-se més de
5.500 trasplantaments hepàtics per any a Europa2,3.
La cirrosi es caracteritza per la formació de septes fibrosos i nòduls de
regeneració, i resulta de diferents mecanismes de lesió hepàtica que causen
necroinflamació, fibrogènesi secundaria a l’activació de les cèl·lules hepàtiques
estrellades, angiogènesi i extinció del parènquima hepàtic. Aquests processos
condueixen a la distorsió del parènquima hepàtic produint un augment de la
resistència vascular hepàtica. A més, també es produeixen uns canvis
funcionals a la microcirculació hepàtica (disfunció endotelial, remodelació
sinusoïdal i formació de shunts intrahepàtics) provocant una disminució de
factors vasodilatadors (òxid nítric, entre altres) i un augment de substàncies
vasoconstrictores (Tromboxà A2, entre altres). L’augment de la resistència
vascular intrahepàtica doncs, es produeix per alteracions en l’arquitectura
hepàtica i per un augment en el to vascular hepàtic, i són el factor inicial en el
desenvolupament de la hipertensió portal. Secundàriament a l’augment de la
resistència vascular intrahepàtica es produeix una vasodilatació arterial de la
circulació esplàncnica que augmenta el flux portal empitjorant i perpetuant la
síndrome de la hipertensió portal4,5.
1.2. Estadis de la cirrosi hepàtica La història natural de la cirrosi comprèn clàssicament diferents fases. Durant la
primera fase el pacient no presenta símptomes i la malaltia es diagnostica de
forma incidental; aquesta fase es coneix com a cirrosi compensada. La segona
13
fase, coneguda com a cirrosi descompensada, es caracteritza per una fase
clínica en la que el pacient desenvolupa complicacions, especialment ascites,
edemes, hiponatrèmia dilucional, i síndrome hepatorenal, així com hemorràgia
digestiva per varices esofàgiques, infeccions bacterianes i encefalopatia
hepàtica6. Existeix una altra classificació que té en compte unes
característiques clíniques i proporciona informació pronòstica (Figura 1).
Aquesta classificació divideix la cirrosi en quatre estadis: 1) Estadi 1: absència
de varices i d’ascites, amb una mortalitat a l’any de l’1%. 2) Estadi 2: varices
esofàgiques sense antecedents d’hemorràgia gastrointestinal per hipertensió
portal ni ascites, amb una mortalitat del 3,4% a l’any. 3) Estadi 3: presència
d’ascites amb o sense varices, amb una mortalitat del 20% a l’any. 4) Estadi 4:
hemorràgia gastrointestinal per hipertensió portal, amb o sense ascites, amb un
57% de mortalitat a l’any7.
Figura 1. Història natural de la cirrosi hepàtica: probabilitat de progressió i mortalitat a 1 any segons l’estadi clínic. Adaptada de D’Amico G et al., J
Hepatol. 2006.
14
Els estadis 1 i 2 corresponen a pacients en fase de cirrosi compensada i els
estadis 3 i 4 a pacients amb cirrosi descompensada6,7. En una proporció no
menyspreable de pacients, la cirrosi pot progressar a una tercera fase en que
es produeix, d’una forma relativament brusca, una descompensació aguda de
la malaltia associada a un fracàs important de les funcions hepàtiques
acompanyat de la disfunció de varis òrgans extrahepàtics, especialment el
sistema cardiocirculatori, els ronyons, el cervell i els pulmons. Aquesta situació
clínica constitueix la síndrome anomenada insuficiència hepàtica aguda sobre
crònica o acute-on-chronic liver failure (ACLF)8.
1.3. Fisiopatologia de la cirrosi
Existeix una àmplia evidència que la vasodilatació arterial perifèrica té un paper
clau en la fisiopatologia de la cirrosi i de les seves complicacions (teoria de la
vasodilatació arterial perifèrica)9–11. En els últims anys, s’ha proposat que la
inflamació sistèmica contribueix també en la fisiopatologia de la cirrosi (teoria
de la inflamació sistèmica)12,13. A la figura 2 es representen gràficament les
dues teories i com es correlacionen l’una amb l’altre i contribueixen en el fracàs
d’òrgans.
1.3.1.
Tal com s’ha comentat anteriorment, els pacients amb cirrosi hepàtica
presenten un augment de la resistència vascular intrahepàtica que provoca un
augment de la pressió en el flux portal, generant hipertensió portal que
condiciona la formació de colaterals porto-sistèmiques i una vasodilatació
arterial de la circulació esplàncnica i sistèmica. Aquesta vasodilatació és
causada per l’alliberació de substàncies vasodilatadores, fonamentalment òxid
nítric, monòxid de carbó i cannabinoids endògens, entre altres, a la circulació
esplàncnica9,14.
Teoria de la vasodilatació arterial
En fases inicials de la malaltia, quan la hipertensió portal és moderada,
l’augment del cabdal cardíac compensa la reducció de la resistència vascular
sistèmica, mantenint la pressió arterial i el volum arterial efectiu. A mesura que
la malaltia progressa, la vasodilatació arterial de la circulació esplàncnica és
molt severa i la resistència vascular sistèmica està marcadament disminuïda.
15
En aquest punt el cabdal cardíac ja no pot compensar-ho, pel que existeix una
disminució del volum arterial efectiu, amb hipotensió arterial15. Aquesta dóna
lloc a l’activació dels baroreceptors, que per tal de mantenir la pressió arterial,
activen els sistemes vasoconstrictors: sistema nerviós simpàtic (SNS), renina-
angiotensina-aldosterona (RAA) i la secreció de vasopressina (AVP). Aquests
mecanismes compensatoris permeten mantenir la pressió arterial relativament
normal, però alhora tenen efectes nocius a nivell renal perquè condueixen a la
retenció de sodi, aigua lliure i la vasoconstricció renal i són els responsables del
desenvolupament de les complicacions de la cirrosi com l’ascites, hiponatrèmia
dilucional o la síndrome hepatorenal, respectivament9,10,14.
1.3.2.
En els últims anys s’ha posat en evidència que la cirrosi ocorre en el context
d’una inflamació sistèmica12,13,16. S’ha demostrat que els pacients amb cirrosi
presenten un augment dels reactants de fase aguda com la proteïna C reactiva
marcadors d’activació macrofàgica22, augment del síndrome de resposta
inflamatòria sistèmica (SIRS)23, entre altres. Aquests marcadors es
correlacionen amb l’estadi de la malaltia i amb el pronòstic. En els pacients
amb ACLF, s’ha observat un augment de la xifra de leucòcits i de la PCR8.
Teoria de la inflamació sistèmica
La hipòtesi actual suggereix que els mecanismes responsables d’aquesta
inflamació sistèmica són16: 1) l’alliberació de components que expressen les
bactèries, els PAMPs (pathogen-associated molecular paterns), que provenen
de la translocació de bactèries viables o dels mateixos PAMPs des de la llum
intestinal a la mucosa. Els PAMPs (p. ex. lipopolisacàrids o flagelina) poden
quedar-se a la mucosa intestinal o ser alliberats a la circulació sistèmica, i tot i
que la majoria de PAMPs són eliminats per les cèl·lules immunitàries
intestinals, en ocasions es produeixen infeccions bacterianes que induirien a
més inflamació24. El segon mecanisme és: 2) l’alliberació de DAMPs
(damage/danger-associated molecular paterns) procedents del dany
hepatocitari, com per exemple heat-shock proteins (HSP) o high-mobility group
protein B1 (HMBG1)16,25,26. Els PAMPs i DAMPs interaccionen amb els
receptors de reconeixement de patògens (PRR; pattern-recognition receptors)
expressats a les cèl·lules immunes innates i en els epitelis. La unió genera
16
l’expressió de gens que codifiquen per la síntesi de molècules implicades en la
inflamació (p. ex. TNF-α, IL-6, etc) tant a la mucosa intestinal com a nivell
hepàtic, i posteriorment poden estendre’s a la circulació sistèmica, generant
una marcada resposta inflamatòria sistèmica25.
Els mediadors inflamatoris alliberats, causarien una major vasodilatació arterial,
empitjorant la disfunció circulatòria existent. A més, els mediadors inflamatoris
podrien causar dany cel·lular i tissular directe provocant una fallida dels òrgans,
similar al que ocorre a la sèpsia13,27.
Figura 2. Fisiopatologia de la cirrosi i de les seves complicacions: teoria de la vasodilatació arterial perifèrica i teoria de la inflamació sistèmica.
17
Figura 2. PAMPs, pathogen-associated molecular paterns; DAMPS, Damaged-
associated molecular paterns; SNS, sistema nerviós simàtic; RAA, renina-
No obstant, la inflamació sistèmica a la cirrosi, ocorre en el context paral·lel
d’una immunodeficiència. La immunodeficiència es produeix per una disminució
de la vigilància immunològica hepàtica, disminució de la síntesis de reactants
de fase aguda i de PRR, i alteració de la funció de les cèl·lules immunitàries. A
més, a mesura que la malaltia progressa, sota una persistent estimulació de les
cèl·lules immunes per PAMPs, la resposta immunitària s’exhaureix, i predomina
el fenotip immunodeficient16,27. Així doncs, la cirrosi presenta una coexistència
dinàmica d’inflamació sistèmica i d’un estat d’immunodeficiència; aquestes
anormalitats del sistema immune que estan presents a la cirrosi es coneixen
com a síndrome de disfunció immune associada a la cirrosi (CADI; cirrhosis-
associated immune disfuntion)16.
1.4. Insuficiència hepàtica aguda sobre crònica o acute-on-chronic liver failure
En els darrers anys s’ha descrit una nova síndrome, la insuficiència hepàtica
aguda sobre crònica o acute-on-chronic liver failure (ACLF), que es va
descriure i definir a partir d’un estudi multicèntric Europeu amb més de 1.000
pacients amb cirrosi descompensada (estudi CANONIC)8. L’ACLF és una
síndrome caracteritzada per una descompensació aguda de la cirrosi,
associada al fracàs d’un o més òrgans i amb una elevada mortalitat a curt
termini8. Els criteris de fracàs orgànic per definir l’ACLF es determinen
mitjançant l’índex SOFA modificat (CLIF-C OF score); (taula 1)8,28. D’acord amb el número i tipus de fracassos orgànics segons l’índex CLIF-C OF
score, es defineix la presència d’ACLF i es classifica segons la gravetat en 3
graus (taula 2).
18
Taula 1. Índex CLIF-C OF score pel diagnòstic d’ACLF.
Òrgan/sistema Subscore=1 Subscore=2 Subscore=3
Fetge, bilirrubina (mg/dL) <6 ≥6 - <12 ≥12
Ronyó, creatinina (mg/dL) <2 ≥2 - <3,5 ≥3,5 o TSR
Cervell, (West-Haven) 0 1 - 2 3-4
Coagulació, (INR) <2,0 ≥2,0 - <2,5 ≥2,5
Circulació, PAM (mmHg) ≥70 <70 Vasoconstrictors
Respiratori
PaO2/FiO2 o >300 o ≤300 i >200 o ≤200 o
SpO2/FiO2 >357 >214 i ≤357 ≤214
L’àrea gris descriu el criteri diagnòstic de fracàs de cada òrgan. *TSR, teràpia de substitució renal; INR; international normalized ratio, PAM Pressió Arterial Mitjana; FiO2, fracció d’oxigen inspirat; PaO2, pressió parcial d’oxigen arterial; SpO2 saturació d’oxigen.
Taula 2. Criteris diagnòstics d’ACLF.
No ACLF Sense fracàs d’òrgan.
1 únic fracàs d’òrgan, excepte fracàs renal, amb una creatinina
sèrica <1,5mg/dL i sense encefalopatia hepàtica.
ACLF grau 1 Fracàs renal únic.
1 únic fracàs d’òrgan associat a disfunció renal (creatinina
entre 1,5 i 1,9 mg/dL) i/o encefalopatia hepàtica grau 1-2.
ACLF grau 2 2 fracassos d’òrgan.
ACLF grau 3 ≥3 fracassos d’òrgan.
La prevalença global de l’ACLF en els pacients hospitalitzats per una
descompensació aguda de la cirrosi és del 30%, i la mortalitat a 28 i a 90 dies
és de 30 i 50%, respectivament. La mortalitat es correlaciona amb el nombre de
fracassos d’òrgans, essent l’ACLF grau 3 el que presenta pitjor pronòstic en
comparació amb els graus 1 o 28.
Els factors precipitants varien segons l’àrea geogràfica, a Europa, les infeccions
bacterianes i l’alcohol són les causes més freqüents. No obstant, en un número
significatiu de pacients (20-40%) no s’aconsegueix identificar cap agent
precipitant29.
19
La patogènia de l’ACLF es desconeix, però hi ha dades indirectes que
suggereixen que ocorre en el context d’una resposta inflamatòria sistèmica. A
l’estudi CANONIC es va observar que els pacients amb ACLF presentaven una
elevació marcada de la PCR i de la xifra de leucòcits, ambdós marcadors
proinflamatoris, que a més es correlacionaven amb el pronòstic8. Hi ha pocs
estudis que avaluïn la inflamació en els pacients amb ACLF, i a més, únicament
determinen un nombre limitat de citocines30,31.
Actualment no existeix cap tractament específic pels pacients amb ACLF. El
maneig consisteix en identificar precoçment la síndrome, tractar els factors
precipitants (infeccions, hepatitis aguda alcohòlica, etc), i el tractament de
suport pels diferents òrgans. El trasplantament hepàtic (TH) és el tractament
definitiu dels pacients amb ACLF, però la utilització del TH en aquests pacients
és complexa ja que en moltes ocasions presenten contraindicacions32,33.
Així doncs, calen estudis per tal d'analitzar la resposta inflamatòria sistèmica
amb diferents tipus de marcadors inflamatoris, així com analitzar les vies
inflamatòries implicades en l’ACLF per tal d’aprofundir en la fisiopatologia i en
les possibles dianes terapèutiques d’aquesta síndrome.
1.5. Insuficiència renal aguda: síndrome hepatorenal La insuficiència renal aguda (AKI, acute kidney injury) és una complicació
freqüent dels pacients amb cirrosi, amb una freqüència del 20-50%
aproximadament dels pacients que ingressen a l’hospital per una complicació
de la malaltia34,35. En els últims anys, el Club Internacional d’Ascites (ICA) ha
canviat la definició de la insuficiència renal a la cirrosi; actualment es basa en
un increment de la creatinina sèrica (sCr) de ≥0,3mg/dL (≥26.5μmol/L) en un
període de 48 hores o un augment de ≥50% de la sCr respecte el valor de la
sCr basal que presumiblement ha ocorregut en els 7 dies previs36. Anteriorment
es definia la insuficiència renal aguda com un augment de la sCr ≥50% des del
basal amb un valor final de >1.5mg/dL37. L’AKI es classifica en diferents estadis
segons la intensitat del canvi de la sCr (taula 3)35,36.
20
Taula 3. Definició i estadis d’AKI segons la classificació ICA-AKI
Definició d’AKI
Augment de la sCr ≥0,3mg/dL (≥26.5μmol/L) en 48h; o augment de la sCr
≥50% a partir del valor basal que presumiblement ha ocorregut en els 7 dies
previs.
Estadis d’AKI
AKI I Augment de la sCr ≥0,3 mg/dL (≥26.5 μmol/L) o augment de la sCr
≥1.5 a 2 vegades respecte el valor basal.
Estadi 1A: sCr al diagnòstic < 1,5mg/dl.
Estadi 1B: sCr al diagnòstic ≥1,5mg/dl.
AKI II Augment de la sCr entre 2 a 3 vegades respecte el valor basal.
AKI III Augment de la sCr més de 3 vegades respecte el valor basal, ò sCr
>4mg/dL (352.6 μmol/L) ò inici de teràpia de substitució renal.
Els pacients amb cirrosi poden presentar insuficiència renal per diferents
causes, però la més característica i única de la cirrosi és la síndrome
hepatorenal10,38.
1.5.1.
Síndrome hepatorenal
La síndrome hepatorenal (SHR) és una forma única d’insuficiència renal que
ocorre en les fases més evolucionades de la cirrosi10,11. Clàssicament la
síndrome hepatorenal es definia en dos subtipus. La SHR tipus 1, que es
caracteritzava per una insuficiència renal ràpidament progressiva amb un
augment del valor de la sCr superior al doble del basal amb una sCr final de
≥2,5mg/dL en un període inferior a dues setmanes; i la SHR tipus 2, que es
caracteritzava per una insuficiència renal moderada, en que la sCr es mantenia
relativament estable durant mesos, amb valors de sCr >1.5mg/dL39. La definició
de la síndrome hepatorenal ha canviat darrerament en el context de la
incorporació dels criteris d’AKI. Els nous criteris de SHR-AKI es presenten a la
taula 436. L’únic canvi entre la nova classificació de SHR-AKI i la definició
21
clàssica de SHR és que s’ha eliminat el punt de tall fix del valor de creatinina
per poder diagnosticar i iniciar el tractament vasoconstrictor de forma més
precoç, i no esperar fins a valors de sCr de >2.5mg/dL per començar el
tractament, amb el que podria determinar una major probabilitat de resposta al
tractament40. Per tant, els pacients amb SHR tipus 1 s’inclouen en la nova
definició de SHR-AKI, però també els pacients que compleixen els criteris d’AKI
i la resta de criteris de SHR, però amb un valor de sCr <2.5mg/dL (non-type 1
SHR). Les característiques clíniques i evolució d’aquest subgrup de pacients
caldrà ser avaluat en futurs estudis.
D’altra banda, el terme SHR tipus 2 ha desaparegut i es considera una forma
d’insuficiència renal crònica (SHR-CKD).
Taula 4. Criteris diagnòstic de la síndrome hepatorenal segons els criteris ICA-AKI.
Criteris diagnòstic de la SHR-AKI
Cirrosi i ascites
Diagnòstic d’AKI segons els criteris de ICA-AKI: augment de la sCr ≥0,3mg/dL
en 48hores
Absència de xoc
Absència de resposta després de 2 dies consecutius de la retirada de diürètics
i expansió amb albúmina (1gr/kg de pes)
Absència d’ús recent de fàrmacs nefrotòxics (antiinflamatoris no esteroïdals,
contrast iodat, aminoglicòsids etc.)
Absència de signes de dany renal estructural, definit com:
- Absència de proteïnúria (>500mg/dia)
- Absència de microhematúria (>50 eritròcits per camp)
- Ecografia renal sense alteracions estructurals
La síndrome es produeix com a conseqüència de la disfunció circulatòria de la
cirrosi que causa una intensa vasoconstricció renal secundària a la
vasodilatació arterial esplàncnica produïda per la hipertensió portal, conduint a
una disminució del filtrat glomerular (figura 2)9,10,41. El tractament d’elecció de la
SHR són els fàrmacs vasoconstrictors, particularment la terlipressina,
22
juntament amb l’albúmina42,43. El tractament té una taxa de resposta del 50%
aproximadament i s’associa a un augment de la supervivència44,45. El fet que el
tractament amb vasoconstrictors millori la funció renal en una proporció
significativa de pacients, confirma el paper de la vasodilatació arterial en la
patogènesis de la síndrome. En tot cas, el tractament definitiu dels pacient amb
SHR és el trasplantament hepàtic, doncs representa la cura de la malaltia
subjacent11.
No obstant, una proporció no menyspreable de pacients no responen al
tractament amb vasoconstrictors, pel que és possible que a part de la disfunció
circulatòria, altres mecanismes estiguin implicats en la fisiopatologia de la
malaltia. Tal com s’ha comentat anteriorment, en els últims anys, s’ha posat de
manifest que la cirrosi s’associa a una inflamació sistèmica i que augmenta
amb la progressió de la malaltia13. La relació entre la SHR i la inflamació s’ha
proposat perquè la presència de SIRS és més freqüent en pacients amb SHR
que en altres causes d’AKI46. També s’ha demostrat que els pacients amb SHR
i peritonitis bacteriana espontània (PBE) presenten valors més alts de IL-6 i
TNF-α que els pacients amb la infecció però sense SHR47. No obstant són
dades limitades i indirectes en condicions específiques de la SHR. Així, no hi
ha estudis que avaluïn la presència, extensió i pronòstic de la inflamació de
forma global i acurada en els pacients amb SHR amb i sense infecció. Donat
que la SHR ocorre en fases evolucionades de la malaltia i comporta una
elevada mortalitat, seria de gran interès obtenir una fotografia completa de la
inflamació per ampliar el coneixement de la patogènesi de les complicacions de
la cirrosi i identificar possibles dianes terapèutiques per prevenir la progressió
de la malaltia.
1.6. Estudi de la inflamació Breument, la resposta inflamatòria aguda causada per una infecció o per dany
tissular implica l’alliberació coordinada de components sanguinis (plasma i
leucòcits) cap al lloc de la infecció o del dany. Aquesta resposta inflamatòria és
desencadenada per la unió de PAMPs i DAMPs amb els receptors PRR que es
troben a les cèl·lules del sistema immune innat. Aquest reconeixement inicial
condueix a la producció de mediadors inflamatoris com quimiocines i citocines,
23
entre altres productes, i l’efecte immediat d’aquests mediadors és generar un
exsudat inflamatori per tal d’eliminar els patògens i el dany48. Per tant,
citocines, quimiocines, factors de creixement endotelial (VEGF) i molècules
d’adhesió endotelial (VCAM-1 i ICAM-1) són claus en el procés de la inflamació
i els hem utilitzat per estudiar la inflamació sistèmica en els dos estudis
d’aquesta Tesi Doctoral. A la taula 5 es presenten de forma resumida tots els
marcadors inflamatoris que s’han determinat, les cèl·lules productores, les
cèl·lules diana i les funcions que realitzen; així com un resum de la informació
més rellevant d’aquests marcadors en els pacients amb cirrosi.
Taula 5. Llistat de marcadors inflamatoris analitzats en els estudis que comprenen aquesta Tesi Doctoral, amb les principals cèl·lules productores, cèl·lules diana i les funcions corresponents; i la informació més rellevant d’aquests marcadors inflamatoris a la cirrosi.
Marcador inflamatori
Pro / Anti-inflamatori
Cèl·lules productores
Dianes cel·lulars i funcions principals
Marcador inflamatori a la cirrosi
TNF-α49,50 Pro Mf, limfòcits, NK
-Cèl·lules (cels) endotelials: activació; promou la inflamació i la coagulació.
-Polimorfonuclears (PMN): activació. -Macròfags (Mf), vàries cels: estimula la síntesi de
citocines (IL-1, IL-6) i quimiocines. -Hipotàlem: febre. -Hepatòcits: síntesi de proteïnes de fase aguda. -Múscul i greix: catabolisme, caquèxia.
-Major concentració de TNF-α a major gravetat de la cirrosi18,20,30,31.
-Pacients amb PBE i SHR presenten més nivells de TNF-α que pacients amb PBE sense SHR47.
INF-γ51 Pro Limfòcits, NK
-Mf: activació clàssica, augment de funcions microbicides.
-Limfòcits B (LB): canvi d’isotip a subclasses de IgG opsonitzadores i fixadores de complement.
-Limfòcit T (LT): diferenciació a TH1. -Altres cels: més expressió de MHC I i II. Major
processament d’antigen i presentació a LT. -Pro-apoptòtic.
-L’INF-γ augmenta la inflamació, dany hepàtic i la fibrosi52.
24
Factors d’estimulació de colònies
G-CSF
Pro
Mf, cels endotelials, fibroblasts
-Cels precursores del moll de l’os: maduració de granulòcits.
-El tractament amb G-CSF millora la supervivència dels pacients amb ACLF53.
GM-CSF54
Pro
Mf, cels endotelials, LT,
fibroblasts
-Cels precursores del moll de l’os: maduració de granulòcits i monòcits.
-Activació, proliferació de Mf, monòcits i PMN.
-GM-CSF pot tenir un paper en la prevenció
d’infeccions de pacients amb cirrosi55.
Interleuquines49,56
IL-1RA Anti Mf -LT, cels endotelials i epitelials: antagonista
competitiu de la IL-1.
IL-1β
Pro
Mf, cels endotelials, hepatòcits, fibroblasts.
-Cels endotelials: activació; promou la inflamació i la coagulació.
-Hipotàlem: febre. -Fetge: síntesi de proteïnes de fase aguda. -LT, cels endotelials: Inducció de citocines/
proteïnes proinflamatòries. -Diferenciació de TH17.
-Pacients amb cirrosi presenten nivells elevats de
IL-1β57.
IL-2 Pro LT, NK
-LT i LB: proliferació i diferenciació en cels efectores i de memòria. Promou el desenvolupament, supervivència i funció de LT reguladors.
-NK: proliferació i activació.
IL-4 Pro
LTH2, mastòcits, eosinòfils, basòfils
-LB: Canvi d’isotip a IgE. -LT: diferenciació i proliferació a TH2. -Mf: activació alternativa. -Adhesió i inflamació tissular.
IL-5 Pro LTH2
-Eosinòfils: activació i augment de l’activitat d’adhesió.
-LB: major generació de LB. Producció de IgA.
IL-6 Pro
Mf, cels endotelials, LT,
fibroblasts.
-Fetge: síntesi de proteïnes de fase aguda. -Leucòcits: activació i mobilització. -LT: diferenciació i activació. -LB: diferenciació i producció de IgG, A, M. -Hematopoesi.
-IL-6 està elevada en pacients amb cirrosi57. Valor elevats d’IL-6 s’associen a major mortalitat58, i amb la gravetat de la cirrosi31.
-Pacients amb PBE i SHR presenten més nivells de IL-6 que pacients amb PBE sense SHR47.
25
IL-7 Pro Cels epitelials,
cels de medul·la òssia
-Progenitors immadurs: proliferació de primers progenitors de LT i LB.
-LT: supervivència de limfòcits verges i de memòria. -Síntesi i inducció de mediadors inflamatoris de
monòcits.
IL-8 o
CXCL8
Pro
Mf, monòcits, PMN, cels, endotelials i
epitelials
Quimiocina: -Quimioatracció de: PMN, NK, LT. -Mobilització de cels mare hematopoètiques. -Angiogènesi.
-Pacients amb cirrosi avançada presenten valors
més elevats de IL-830,31.
IL-9 Pro LTH2, mastòcits
-Mastòcits, LT CD8+, LB i cels tissulars: supervivència i activació.
-Producció de IgE.
IL-10 Anti Mf, monòcits, LT, LB
-Immunosupressió. -Mf, cels dendrítiques: inhibició de IL-12, de
coestimuladors i de MHC tipus 2.
-Pacients amb ACLF presenten valors més elevats
de IL-10 que pacient amb cirrosi sense ACLF30,31.
IL-12 (P70) Pro Mf, cel
dendrítica, PMN
-LT CD4+: diferenciació a TH1. -NK i LT CD8+: síntesi de INF-γ i augment de
l’activitat citotòxica.
IL-13 Pro LTH2,
-LB: Canvi d’isotip a IgE. -Cels epitelials: major producció de moc -Fibroblasts: síntesi de col·lagen. -Mf: activació alternativa. -Eosinòfils i mastòcits (protecció d’infeccions
parasitàries).
IL-15 Pro Mf
-NK: proliferació. -LT: supervivència i proliferació de CD8+ de
memòria.
IL-17A Pro LT; LTH17
-Cels endotelials/epitelials i Mf: major producció de quimiocines, citocines, GM-CSF i G-CSF.
Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with PrognosisCristina Solé1,2,3, Elsa Solà1,2,3, Manuel Morales-Ruiz2,3,4, Guerau Fernàndez5, Patricia Huelin1,2,3, Isabel Graupera1,2,3, Rebeca Moreira1,2,3, Gloria de Prada1,2,3, Xavier Ariza1,2,3, Elisa Pose1,2,3, Núria Fabrellas2,6, Susana G. Kalko5, Wladimiro Jiménez2,3,4 & Pere Ginès1,2,3
ACLF is characterized by a systemic inflammatory response, but the cytokines involved in this process have not been well studied. The aim of this study was to characterize the systemic inflammatory response in patients with cirrhosis and ACLF and its relationship with prognosis. Fifty-five patients with cirrhosis, 26 with ACLF, were studied prospectively. Systemic inflammatory response was analyzed by measuring a large array of plasma cytokines by using a multiplex kit. A principal component analysis show noticeable differences between ACLF and decompensated cirrhosis without ACLF. Patients with ACLF had significant abnormal levels of 12 cytokines compared to those without ACLF, including: VCAM-1, VEGF-A, Fractalkine, MIP-1α, Eotaxin, IP-10, RANTES, GM-CSF, IL-1β, IL-2, ICAM-1, and MCP-1. Cytokines showing the most marked relationship with ACLF were VCAM-1 and VEGF-A (AUCROC 0.77; p = 0.001). There was a significant relationship between some of inflammatory mediators and 3-month mortality, particularly VCAM-1, ICAM-1, and GM-CSF (AUCROC>0.7; p < 0.05). Functional Enrichment Analysis showed that inflammatory markers differentially expressed in ACLF patients were enriched in leukocyte migration, particularly monocytes and macrophages, and chemotaxis pathways. In conclusion, ACLF is characterized by a marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes. The intensity of the inflammatory reaction correlates with prognosis.
Liver cirrhosis is a chronic disease characterized by relentless deposition of collagen and disruption of the normal liver architecture that causes progressive portal hypertension and liver failure that eventually leads to complica-tions and death unless liver transplantation is performed1. There is increasing evidence supporting the existence of a systemic inflammatory reaction in cirrhosis that contributes to complications and disease progression2,3. This systemic inflammatory reaction is likely initiated by translocation of bacteria or bacterial products from the intes-tinal lumen to the mesenteric lymph nodes and then reaching the systemic circulation. This leads to increased levels of pathogen-associated molecular patterns (PAMPs) that stimulate pattern recognition receptors (PRRs), expressed on innate immune cells. Moreover, the generation of damage-associated molecular patterns (DAMPs) from the diseased liver may also stimulate immune cells. Once stimulated, PRRs induce a transcriptional response leading to synthesis of a number of pro and anti-inflammatory cytokines, chemokines, cell adhesion molecules responsible for an adaptive immune response4,5. It has been shown that treatments that reduce bacterial trans-location reduce the intensity of the immune response, while a number of complications, particularly bacterial infections are associated with an increased intensity of the immune reaction6,7. It is currently believed that such
1Liver Unit, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spain. 2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. 3Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEReHD), Barcelona, Spain. 4Biochemistry and Molecular Genetics Department, Hospital Clínic de Barcelona, Barcelona, Spain. 5Bioinformatics Core Facility, IDIBAPS-CEK, Hospital Clínic, University de Barcelona, Spain. 6Facultat de Medicina i Ciències de la Salut, University of Barcelona, Barcelona, Spain. Correspondence and requests for materials should be addressed to E.S. (email: [email protected])
“chronic” inflammatory response may lead to a paralysis of the immune system, which in turn may be pathogen-ically related to the high frequency of severe infections that occur in patients with cirrhosis8,9.
There is growing interest among clinicians and researchers about acute-on-chronic liver failure (ACLF), a syndrome that occurs in patients with chronic liver diseases, particularly cirrhosis, which is characterized by development of failure of different organs and systems and high mortality rate10,11. The hypothesis has been raised that ACLF is associated with a remarkable inflammatory state that contributes to the pathogenesis and progres-sion of this syndrome10,12. This hypothesis is based on the findings of increased leukocyte count and C-reactive protein (CRP) levels in patients with ACLF compared to those of patients with cirrhosis without ACLF and their correlation with prognosis13. Yet bacterial infections are very common as precipitating events of ACLF, it has been suggested that the inflammatory reaction in ACLF may occur in the absence of bacterial infections, at least undetectable by current standard diagnostic methods10,13. Nonetheless, despite these suggestive findings, there is very little information on the type of inflammatory mediators that are increased in ACLF and its relationship with outcomes. Moreover, the few studies published have investigated only either single or limited number of cytokines9,14; therefore neither a complete picture about the characteristics of the inflammatory reaction nor the types of pathways involved are known. Therefore, the current study was aimed at addressing the issue of the inflammatory response and its relationship with ACLF and survival in patients with cirrhosis. A large number of cytokines was measured in patients with and without ACLF using a multiplex approach. Moreover, the results were analyzed with a principal component analysis and functional enrichment analysis to gain further insight on activated inflammatory pathways. Our findings demonstrate that the syndrome of ACLF is characterized by marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes, particularly monocytes and macrophages. Moreover, the levels of some of these cytokines are associated with prognosis, a finding that links the inflammatory reaction with outcome in ACLF.
ResultsBaseline characteristics of patients. The current study includes 55 patients with decompensated cirrho-sis, 26 with ACLF and 29 without ACLF, admitted to the Liver Unit of the Hospital Clínic in Barcelona for the management of complications of the disease. Demographic, clinical, and analytical data were collected prospec-tively at admission and during hospitalization. Blood samples for the measurement of cytokines were collected at the time of inclusion in the study. The characteristics of patients at time of inclusion in the study are shown in Table 1. As expected, patients with ACLF had greater frequency of ascites, hepatic encephalopathy, and shock compared to that of patients with acute decompensation of cirrhosis without ACLF. Moreover, liver function tests, Child-Pugh score and model of end-stage liver disease (MELD) score, serum creatinine, serum sodium, and mean arterial pressure were more markedly impaired in patients with ACLF than in those without ACLF. Of interest, neither the frequency of bacterial infections nor that of systemic inflammatory response syndrome (SIRS) was significantly different among groups. Leukocyte count and CRP levels were higher in patients with ACLF but the difference did not reach statistical significance.
Cytokine levels and relationship with ACLF. An initial exploration of the concentrations of cytokines was performed using principal component analysis (PCA) including all cytokines with and without standard laboratory variables in all cirrhotic patients as well as in healthy subjects. Figure 1A shows a three dimensional scatter plot corresponding to the first three principal components including only cytokines. A relatively good distinction between healthy subjects, patients with acute decompensation of cirrhosis without ACLF, and patients with ACLF was observed. The group of healthy subjects was clearly separated from the other two groups. The group of patients with ACLF was scattered at the opposing end of healthy subjects, whereas patients with acute decompensation of cirrhosis without ACLF had a more heterogeneous distribution and some of them overlapped with patients with ACLF. A slightly better distinction between the 3 groups was observed when laboratory varia-bles were added to cytokines in the principal component analysis (Fig. 1B).
Table 2 shows the levels of different cytokines in patients with and without ACLF with their respective AUCROC curves. Patients with ACLF had significantly increased levels of vascular cell adhesion molecule 1 (VCAM-1), vascular endothelial growth factor A (VEGF-A), fractalkine, macrophage inflammatory protein 1-aplha (MIP-1α ), eotaxin, and interferon-inducible protein-10 (IP-10) compared to those of patients without ACLF. Levels of intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) were also higher in patients with ACLF but did not reach statistical significance. By contrast, levels of RANTES (Regulated on activation, normal T cell expressed and secreted), Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 beta (IL-1β ), and interleukin 2 (IL-2) were significantly lower in patients with ACLF compared to those of patients without ACLF. There were no significant differences in the levels of the remaining cytokines.
Figure 2 shows the individual values of cytokines showing statistical significance (including the two cytokines with p values between 0.05 and 0.10) in patients with and without ACLF. A group of healthy subjects was also included for comparison. Two main messages can be derived from a close observation of this figure. First, there was overlap in the levels of most cytokines between patients with and without ACLF. Second, despite this overlap there was a clearly progressive increase (or decrease) of median cytokine levels from healthy subjects to patients without ACLF and patients with ACLF, suggesting that disease progression from decompensated cirrhosis with-out ACLF to ACLF is associated with significant changes in cytokine pattern. Pathway enrichment analysis using cytokines that were statistically different among groups showed that the most significant functional terms were related to migration and chemotaxis of leukocytes. Interestingly, most of the pathways were related to monocytes and macrophages (Table 3).
To assess whether the abnormal cytokine pattern found in patients with ACLF could be related to presence of bacterial infections, we next compared cytokine levels in patients with ACLF with those of the subset of patients
without ACLF but with bacterial infections. The cytokines that showed statistically significant difference between the two groups of patients were the same cytokines which were significantly altered in ACLF vs no ACLF, with the only exception of VEGF-A, which showed a trend towards statistical significance (p = 0.079) (Table 4). These findings suggest that the abnormal cytokine pattern of ACLF does not appear to be related, at least for the most part, to the presence of bacterial infections.
Cytokine levels and relationship with survival. At the end of the 3-month follow-up period, 15 patients (27%) had died, 3 (5%) had been transplanted, and the remaining 37 patients (67%) were still alive. Twelve of the 26 patients from the ACLF group died compared to only 3 of the 29 patients without ACLF (46% vs 10%, p < 0.001). The probability of survival of patients according to the presence or absence of ACLF is shown in Supplementary Figure 1. ACLF was the cause of death in all patients from the ACLF group, whereas the 3 patients from the no ACLF group died due to septic shock (two patients) and ACLF. The 3 patients transplanted belonged to the ACLF group.
Several cytokines showed an association with 3-month mortality. The cytokines with strongest association with mortality (AUCROC > 0.7) were VCAM-1, ICAM-1, and GM-CSF (Table 5 and Fig. 3). Increased levels of VCAM-1 and ICAM-1 were associated with increased mortality rate. By contrast, reduced levels of GM-CSF were associated with high mortality. In multivariate analysis including these 3 cytokines, only VCAM-1 was associated with independent prognostic value (Supplementary Table 1). The independent prognostic value of VCAM-1 was maintained even when individual variables known to have a powerful prognostic value, such as leukocyte count or bilirubin levels, were included in the multivariate analysis.
DiscussionThe findings of the current study indicate that ACLF syndrome is associated with an abnormal plasma cytokine profile, characterized by alterations of cytokines mainly related to chemotaxis and migration of leukocytes, par-ticularly monocytes and macrophages. These findings confirm the existence of a marked inflammatory reaction in the setting of ACLF. The abnormal plasma cytokine profile is already present in patients with decompensated cirrhosis but is markedly enhanced in patients with ACLF. Moreover, some of the cytokines correlated with prog-nosis, a finding that links the inflammatory activity with disease outcome.
The landmark CANONIC study shed light on the existence and clinical relevance of systemic inflammation in decompensated cirrhosis and ACLF syndrome. Main findings of this study were: 1/ systemic inflammation, as assessed by leukocyte count and CRP levels, seems to be an important pathogenic component of the ACLF syn-drome; 2/ systemic inflammation in ACLF is independent of the existence of bacterial infections; and 3/ systemic
All patients n = 55Acute decompensation without ACLF n = 29 ACLF n = 26 P*
Age (yr) 60 (51–68) 61 (52–72) 57 (51–65) 0.235
Male sex 42 (76) 22 (76) 20 (77) 0.926
Alcoholic cirrhosis 27 (49) 13 (45) 14 (54) 0.504
Presence of ascites 44 (80) 19 (66) 25 (96) 0.005
Presence of encephalopathy 14 (26) 2 (7) 12 (46) 0.001
Table 1. Demographic, clinical, and laboratory characteristics of patients included in the study. SIRS, systemic inflammatory response syndrome; INR, international normalized ratio; MELD, model for end-stage liver disease; CLIF-C ACLF score (CLIF Consortium ACLF score), CLIF-C AD score (CLIF Consortium Acute Decompensation score). Values are expressed as numbers (%) or median and IQR (in brackets). *Comparison between Acute decompensation without ACLF vs ACLF group.
inflammation is associated with poor short-term mortality13. There is very little information about inflamma-tory cytokines in ACLF. Earlier studies showed increased serum levels of interleukin 6 (IL-6) and interleukin 10 (IL-10) in patients with ACLF compared to those of patients with stable cirrhosis, in a manner similar to that reported in patients with severe sepsis8. However, this study used a definition of ACLF different from the cur-rently accepted definition. A recently published study, which investigated the expression of MERTK in immune cells showed greater serum levels of tumor necrosis factor-alpha (TNF-α ), IL-6, IL-10, and interleukin 8 (IL-8) in patients with ACLF than in those without ACLF, but normal levels of Interferon gamma (IFN-ϒ ), transforming growth factor beta 1 (TGF-β 1), IL-1β , and interleukin 12 (IL-12)9, suggesting the existence of an altered cytokine profile in ACLF. The current study extends these observations by assessing a larger number of cytokines, using a multiplex approach, in a series of patients without ACLF and with well-defined ACLF. Our findings indicate that in decompensated cirrhosis without ACLF there is already an abnormal plasma cytokine profile compared to healthy subjects, which is further stressed in the setting of ACLF. The presence of abnormal plasma cytokine profile was suggested by using PCA approach and demonstrated by standard comparison of cytokine levels among groups (see Figs 1 and 2). An interesting observation of our study is that this abnormal plasma cytokine profile is not exclu-sively related to bacterial infections, because significant differences in cytokine levels persisted among groups when patients with ACLF were compared to the subset of patients with associated bacterial infections without ACLF.
A striking finding of our investigation was that most of the cytokines altered in patients with ACLF were related functionally with chemotaxis and migration of leukocytes, particularly monocytes and macrophages. This finding points towards an important role of monocytes/macrophages in the pathogenesis of ACLF. This observation is in keeping with previous studies showing impaired “sepsis-like” monocyte function in patients with ACLF. Wasmuth et al. showed that patients with ACLF have abnormal monocyte function as indicated by impaired ex-vivo produc-tion of TNF-α after stimulation with lipopolysaccharide (LPS) and reduced HLA-DR expression, suggesting the existence of a functional impairment of the innate immune response8. More recently, it has been shown that ACLF is characterized by an increased number of monocytes and macrophages that express MERTK in circulation, liver, and lymph nodes, compared to patients without ACLF and healthy controls, which correlated with inflammatory response. These MERTK-positive immune cells have an impaired response to LPS stimulation and may likely contribute to ACLF progression and infectious complications9. Along the same lines, our study showed that ACLF was associated with reduced levels of GM-CSF. Moreover, reduced levels of GM-CSF correlated with mortality, further suggesting the important role of the monocyte/macrophage system in determining poor outcome in cir-rhosis. GM-CSF is member of a family of hematopoietic growth factors that mobilize immune cells from bone marrow and also enhances the activity of different types of leukocytes, including neutrophils and monocytes15,16. To our knowledge, there are no data on GM-CSF levels in cirrhosis and its relationship with ACLF and outcome. Interestingly, a recent study in patients and animals with experimental acute liver failure (ALF) showed that ALF is associated with reduced levels of CSF1 (also known as macrophage colony-stimulating factor, M-CSF), a growth factor that stimulates monocytes exclusively, which predicted poor outcome17. In the same study, it was shown that administration of CSF1 to animals with ALF improved liver regeneration. These findings are in keeping with recent studies showing that the administration of granulocyte colony-stimulating factor (G-CSF) reduces bacterial infections and improves survival in patients with decompensated cirrhosis without ACLF as well as in ACLF18,19. Although these latter studies include a small number of patients and require confirmation in larger series, overall, these data point towards an important role of impaired monocytes/macrophages function in the pathogenesis of ACLF. Further studies are needed to investigate the cause of impaired monocytes/macrophages, as well as neutro-phils, function and whether colony-stimulating factors could be an effective approach to therapy of ACLF.
Figure 1. Principal Component Analysis (PCA) of all subjects included in the study with only cytokine data (Panel A) or cytokine and biochemical data (Panel B). Confidence region (95%) was indicated by an ellipsoid for each group. Each circle corresponds to one patient. Blue circles: healthy subjects; green circles: patients with acute decompensation without ACLF; Red circles: patients with ACLF.
Another interesting observation of the current study was the markedly increased levels of VCAM-1 and ICAM-1 in patients with ACLF and their relationship with survival, so that higher levels were associated with reduced survival. VCAM-1 and ICAM-1 are molecules that are expressed in the endothelial cells, particularly in post-capillary venules, but also liver sinusoidal cells, that participate in the slow rolling, arrest and adhesion, crawling, transmigration, and diapedesis of leukocytes20,21. Because all these processes are essential initial steps in inflammation, these findings underscore the relevance of leukocyte chemotaxis and migration in ACLF and its relationship with prognosis. Similar findings of increased VCAM-1 and ICAM-1 levels have been observed in sepsis and correlate with prognosis in this condition22. Moreover, increased levels of ICAM-1 and VCAM-1 have been reported in patients with decompensated cirrhosis, particularly those with advanced liver failure, that correlated with prognosis23,24.
Because VCAM and ICAM are produced in endothelial cells, increased levels of these proteins have also been used as surrogate markers of endothelial dysfunction in sepsis as well as in cardiovascular diseases25–27. Therefore, our findings support the existence of endothelial dysfunction in ACLF.
It should be noted that despite that our study investigated a wide range of inflammatory mediators, there are still other potential interesting molecules (i.e., cytokines, chemokines) that could not be investigated as the number and type of measured molecules was limited to the multiplex kit selected. In this context, two recent studies have investigated the role of CXCL9 and CXCL11 in patients with cirrhosis receiving TIPS. Interestingly, results of these studies showed that increased levels of CXCL9 (monokine induced by human gamma interferon) and CXCL11 (Interferon-inducible T-cell alpha chemoattractant) were independent predictors of mortality in patients with cirrhosis receiving TIPS28,29. Therefore, it would be interesting to further investigate the role of these chemokines in the setting of ACLF in future studies.
The current study has some limitations that should be mentioned. First, the sample size is relatively low. However, patients included belong to two clearly differentiated clinical phenotypes of patients with and with-out ACLF, which makes the comparison between the two groups very accurate by avoiding clinical overlap. No patient without ACLF was in the process of rapid development of ACLF, which could have been a confounding factor. In fact, none of the patients without ACLF developed ACLF within one month after inclusion in the study. With respect to patients with ACLF, their characteristics were very similar to those of patients in the CANONIC study, with similar ACLF grades (50%, 31% and 19% of patients with grades I, II, and III in the current study vs 49%, 36%, and 16%, respectively, in the CANONIC study) and mortality (28-day and 90-day mortality of 27% and 46% in the current study vs 34% and 51%, respectively, in the CANONIC study). Second, plasma cytokine levels were measured exclusively at inclusion in the study; a second measurement at a later time point was not available. Therefore, assessment of changes in plasma cytokine levels according to certain outcomes, such as improvement/worsening of ACLF could not be made. However, plasma cytokines levels correlated with mortality which is the most important clinical outcome in ACLF. Finally, the use of a multiplex system allows the measure-ment of a large number of cytokines but is associated with some intrinsic limitations. The main limitation is that some of the cytokines included in the system could not be measured. This is probably related to cross-reading that
CytokineAcute decompensation without ACLF n = 29 ACLF n = 26 P AUCROC (95% IC)
Table 2. Comparison of plasma cytokine levels between patients with acute decompensation of cirrhosis without ALCF and patients with ACLF. AUCROC, area under the receiver-operating characteristic curves. Values are expressed as median and IQR (in brackets).
may affect negatively the sensitivity of detection. This may explain why some cytokines that have been reported as increased in cirrhosis (i.e., TNF-α , IL-6) were not detected by the method used in the current study6,30.
In conclusion, the results of the current study show that the syndrome of ACLF is characterized by marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes, particularly mono-cytes and macrophages. The intensity of the inflammatory reaction correlates with prognosis.
MethodsPatient population and study design. The current study includes 55 patients with decompensated cir-rhosis, 26 with ACLF and 29 without ACLF, admitted to the Liver Unit to the Hospital Clínic in Barcelona for the management of complications of the disease. These patients were selected from a prospective database with biobank collection that includes consecutive patients with decompensated cirrhosis admitted to hospital for treat-ment of an acute decompensation of the disease. Patients were randomly selected from the database from the groups with or without ACLF. Exclusion criteria were: previous kidney/liver transplantation, chronic haemodial-ysis before admission, hepatocellular carcinoma outside the Milan criteria or any other advanced malignancy and lack of inform consent. Causes of admission in patients without ACLF were: infection, ascites, gastrointestinal bleeding, and hepatic encephalopathy (13, 10, 4, and 2 patients, respectively). Importantly, none of the patients without ACLF developed ACLF during at least one month after sample collection. All patients with ACLF met the criteria of ACLF at admission to hospital. Causes of admission in patients with ACLF were: acute kidney injury, infection, ascites, gastrointestinal bleeding, and hepatic encephalopathy (9, 6, 4, 3, and 4 patients, respectively). A group of healthy subjects was included for comparison of plasma cytokine levels.
Demographic, clinical, and analytical data were collected prospectively at admission and during hospitali-zation. All complications developing during hospitalization were recorded and managed according to protocols of the Liver Unit which are based on international treatment guidelines31,32. ACLF was defined according to the CANONIC study13. Patients discharged from hospital were followed-up for at least 3 months.
Figure 2. Individual values of plasma cytokines levels in healthy subjects (HS), patients with acute decompensation of cirrhosis without ACLF (AD) and patients with ACLF (ACLF). Cytokine levels are expressed in log scale.
Blood samples were collected at the time of inclusion in the study. The median time between admission to hospital and collection of samples was 1 day (0 to 3 days). Blood was centrifuged at 2,000G, at 4 °C, for 10 minutes. Plasma was stored at − 80 °C until analysis. All samples were stored at the biobank as required by spanish law. All patients signed a written informed consent document and gave permission for samples to be used in the study following current national and institutional guidelines for sample storage and usage for research purposes. All the analysis and the sample collection were performed in accordance with relevant guidelines and regulations. This study was presented and approved by the Ethics Committee of Hospital Clinic registration number 2014/0577. The creation of the biobank collection was presented and approved by the Ethics Committee registration number (2011/6689).
Enriched processes/pathways Num Cytokines involved Types of cytokines P
Cell movement of monocytes 9 Eotaxin, MIP-1α , RANTES, GM-CSF, Fractalkine, IP-10, IL1-β ,IL-2,VCAM-1 8.44e−20
Table 3. Pathway enrichment analysis revealing specific immune inflammatory response pathways involved in acute-on chronic liver failure. Only the most significant enriched biological processes and pathways based on the p value are shown in the table. The number of cytokines (Num) and types of cytokines overlapping with the significant pathways are also shown.
Table 4. Comparison of plasma cytokine levels between patients with acute decompensation of cirrhosis without ACLF with associated bacterial infections and patients with ACLF. Values are expressed as median and IQR (in brakets).
Multiplex cytokine assay. The following 34 cytokines and growth factors were determined with the Procarta® Immunoassay Kit (Panomics, Affymetrix Inc., Santa Clara, USA): IFN-γ , fibroblast growth factor (FGF-2), interleukin-1 receptor antagonist (IL-1RA), GM-CSF, Interleukin 4 (IL-4), IL-6, IL-8, IP-10, MIP-1α , mac-rophage inflammatory protein 1-beta (MIP-1β ), platelet-derived growth factor subunit B (PDGF-BB), TNF-α , eotaxin, IL-1β , IL-2, interleukin 5 (IL-5), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12p70), interleukin 27 (IL-27), interleukin 21 (IL-21), interleukin 15 (IL-15), MCP-1, placental growth factor-1 (PIGF-1), VEGF-A, interleukin 23 (IL-23), interleukin 17 (IL-17A), interleukin 13 (IL-13), IL-10, fractalkine, G-CSF, RANTES, VCAM-1 and ICAM-1. Briefly, 50 μ L of microparticles precoated with specific antibodies were added to each well with standards or 25 μ L of plasma samples and incubated for 60 minutes at room temperature in the dark. After washing the plate, 25 μ L of detection antibody solution was added and the plate was incubated for 30 minutes at room temperature in the dark. A mix with streptavidin-PE solution was added to the plate for 30 minutes and then the median relative fluorescence units from the antibody reactions was measured in 120 μ L of reading buffer using a Luminex 200 analyzer (Luminex, Austin, TX, USA) and the xPONENT software (v. 3.1; Luminex, Austin, TX, USA). The concentration of each analyte was calculated using five-parameter regression models. We only considered standard points with recoveries ranging from 70 to 130%. The intra-assay coefficient of variation was less than 8%. In individual samples with values below the detection limits, the lower level of detection was used in the calculation of the results. Cytokines in which the levels were below the detection limit in more than 30% of the samples (n = 14) were excluded from the analysis (the median number of samples below detection limits in this subset was 69%). These cytokines were: FGF-2, IL-1RA, IL-4, IL-8, TNF-α , IL-5, IL-9, IL-12p70, IL-27, IL-21, IL-15, PIGF-1, IL-23, and IL-17A.
Principal component analysis. This is a technique developed for simplifying a dataset, based on orthogo-nal linear transformations, that converts the data to a new coordinate system such that the greatest variance by any projection of the data comes to lie on the first coordinate (first principal component), the second greatest variance on the second coordinate, and so on33. By this way, it may be determined whether the variation of protein abun-dance represents defined patterns that correlate with the already defined sample groups. We performed PCA of the integration of multiplex cytokines abundance and other biochemical data in R platform (www.r-project.org) using “stats” and “rgl” packages. An ellipsoid from the covariance matrix was drawn to show the confidence region (95%) for each of the three plotted groups.
Statistical analysis. Categorical variables were compared with the Chi-Square test. Comparisons of par-ametric continuous variables between groups were made with Student’s T-test or ANOVA. Comparisons of non-parametric continuous variables between groups were made with Mann-Whitney U or Kruskal-Wallis tests. Alpha error was adjusted according to Bonferroni correction method in multiple comparisons. The area under the receiver-operating characteristic curves (AUCROC) was used to assess the relationship between each cytokine and outcomes, specifically: ACLF and 90-day transplant-free survival. Survival probability curves were calculated with the Kaplan-Meier method and compared with log-rank test. Multivariate Cox regression was performed to
Variables Alive (n = 37) Dead (n = 15) P Mortality AUCROC (95% IC)
Table 5. Comparison of plasma cytokine levels in patients included in the study categorized according to 3-month survival. AUCROC, area under the receiver-operating characteristic curves. Values are expressed as median and IQR (in brackets). The 3 patients transplanted during the 3-month follow-up period were excluded from the analysis.
identify the independent factors associated with mortality. All statistical analyses were performed using SPSS 20.0 software. Results for continuous variables are expressed as median and interquartile range (IQR). Categorical variables are expressed as number and percentage. The significance level for all tests was set at 0.05 two-tailed.
Functional enrichment analysis. Functional analysis was analyzed through the use of QIAGEN’s Ingenuity Pathway Analysis (IPA® , QIAGEN Redwood City, www.qiagen.com/ingenuity). The significance value associated with the functional enrichment analysis for a given dataset is a measure of the likelihood that the asso-ciation between a set of focus molecules in the experiment and a given process or pathway is not due to random chance. The p-value is calculated using the right-tailed Fisher Exact Test. We selected 1.0e−07 as the significance threshold.
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AcknowledgementsThis study was funded by a grant awarded to P.G. (PI12/00330), from Fondos de Investigación de Salud Carlos III integrated in the Plan Nacional I + D + I and co-funded by ISCIII-Subdirección General de Evaluación and European Regional Development Fund FEDER. Cristina Solé was supported by a grant from the Instituto de Salud Carlos III (PFIS fellowship: FI14/00227). Isabel Graupera was supported by a grant from the Plan Estatal I + D + I and Instituto Carlos III (Rio-Hortega fellowship: CM14/00122). Patricia Huelin was supported by a grant from the University of Barcelona (APIF2015). P.G. is a recipient of an ICREA Academia Award and AGAUR (Agencia de Gestió d’Ajuts Universitaris I de Recerca) Award (2014/SGR 708). We thank Nicki Van Berckel for the administrative support during all the process of this project. We also thank the medical and nursing staff of the liver unit for their participation and the patients who participated in the study and their families.
Author ContributionsAll authors have contributed to this manuscript and approve the version of this submission. C.S. contributed to the conception and design of the study, acquisition of data, the analysis and interpretation of the data and drafting the manuscript; M.M.-R., G.F., P.H., I.G., R.M., G.d.P., X.A., E.P., N.F., S.G.K. and W.J. participated in the generation and collection of data, assembly of data, analyses of the results, interpretation of data, and/or critical revision of the manuscript for important intellectual content. P.G. and E.S. participated in the study concept, interpretation of the data, drafting the manuscript, critical revision of the manuscript for important intellectual content, obtained funding and study supervision.
Additional InformationSupplementary information accompanies this paper at http://www.nature.com/srepCompeting financial interests: The authors declare no competing financial interests.How to cite this article: Solé, C. et al. Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with Prognosis. Sci. Rep. 6, 32341; doi: 10.1038/srep32341 (2016).
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license,
A large body of evidence demonstrating that circulatory dys‐functionplaysakey role in thepathophysiologyofHRS‐AKI.2‐4 Ithas been shown thatHRS‐AKI occurs as a consequence of an in‐tense renalvasoconstrictionwithmarkedreduction in renalbloodflow and glomerular filtration rate (GFR), which is pathogenicallyrelated to a striking splanchnic arterial vasodilatationwith activa‐tionofmajorvasoconstrictorsystems,asaconsequenceofportalhypertension.4,5 Administration of vasoconstrictors, particularlyterlipressin,inassociationwithalbumin,improvescirculatoryfunc‐tionandisabletoreturnrenalfunctiontobaselinevaluesinmanypatients,which confirms themajor roleof circulatorydysfunctionwitharterialvasodilationinthepathogenesisofkidneyimpairmentinHRS‐AKI.6,7
Inrecentyears,ithasbecomeincreasinglyevidentthatcirrhosisisaconditionwithmarkedsystemicinflammatorystate,whichappearstoincreasewithdiseaseprogression,fromcompensatedtodecom‐pensatedcirrhosis,andisrelatedtopatientoutcome.8,9Someclinicalstudieshaveassessed inflammationbymeasuring leucocytecountorC‐reactiveprotein(CRP)levels,10whileothershaveassessedtheprevalence of systemic inflammatory response syndrome (SIRS)11; however,thesemethodsarenotveryaccurateintheevaluationofinflammation. Besides, studies in experimental animals have alsodemonstratedtheexistenceofsystemic inflammationthat ismoreconspicuous in animalswith ascites compared to thosewithout.12 Finally,fewstudieshaveevaluatedalargenumberofinflammatorycytokinesandconfirmedthepresenceofsystemicinflammation,asestimatedbyincreasedplasmalevelsofrelevantcytokinesinvolvedininflammation,whichincreasewithdiseaseprogression.8,13,14Thehypothesis, therefore, has been raised that cirrhosis is a diseasecharacterizedbymarkedandprogressivesystemicinflammationthatmayplayaroleinthedevelopmentofcomplicationsofthedisease.15 However,sofarthereislackofinformationregardingthepresence,
Onthisbackground,theaimofourstudywastoassesstheex‐istenceofsystemicinflammation,asestimatedbyplasmalevelsofalargenumberof inflammatorycytokines, inpatientswithHRS‐AKIand investigate the relationshipbetween inflammationandkidneyandpatientoutcomes.BecausesystemicinflammationiscommoninACLFandpatientswithHRS‐AKIfrequentlymeetdiagnosticcriteriaofACLF,theroleofthislatterconditionaspotentialcauseofinflam‐mationinHRS‐AKIwasalsoassessed.
2 | PATIENTS AND METHODS
2.1 | Patient population and study design
One‐hundred and sixty‐five episodes of AKI occurring in 161 pa‐tientswithdecompensatedcirrhosisadmitted to theLiverUnitofHospitalClinicwereinvestigatedinthecurrentstudy.ThesepatientswereselectedfromaprospectivedatabasewithBiobankcollection
• A systems biology analysis approach showed that theinflammatorystatusofhepatorenalsyndromewassimi‐lar to that found in chronic nonhepatic inflammatorydiseases,suchaslupuserythematosusorinflammatorybowel disease.
| 3SOLÉ et aL.
thatincludesconsecutivepatientswithcirrhosisadmittedtohospi‐talfortreatmentofanacutedecompensationofthedisease.Threegroupsofsubjectswereidentifiedrandomlyfromthedatabase:(1)patientswithdecompensatedcirrhosiswithoutAKI(N=44),(2)pa‐tientswithhypovolaemia‐inducedAKI(N=63)and(3)patientswithHRS‐AKI(N=58).Forty‐oneofthe58patients(71%)withHRS‐AKImet the classical criteria of type‐1 HRS.16 Exclusion criteria werepreviouskidney/livertransplantation,chronichaemodialysisbeforeadmission,hepatocellularcarcinomaoutsidetheMilancriteriaoranyotheradvancedmalignancy,andlackofinformconsent.
Demographical, clinical and analytical datawere collected pro‐spectivelyatadmissionandatregularintervalsduringhospitalization,andpatientswerefollowedupforatleast3monthsafterdischarge.Bloodandurinesampleswerecollectedatthetimeofinclusioninthisstudy.Moreover,in27patientswithtype1‐HRS,sampleswerealsocollectedaftertreatmentwithterlipressinandalbumin(mediantimebetweenthetwosamplecollectionswasof7days).Bloodwascentri‐fugedat2000g,at4°C,for10min,andplasmawasstoredat−80°Cuntil analysis. All samples were stored at the Biobank as requiredbySpanishlegislation.ThisstudywasapprovedbytheInstitutionalReviewBoard of our centre, the research Ethic Committee of theHospitalClínicofBarcelona(HCB/2017/0285),andallpatientssignedawritteninformedconsentforparticipationinthisstudy.
2.2 | Multiplex cytokine assay
The following 18 cytokines and vascular adhesion moleculeswere determined in plasma with the Luminex® Immunoassay Kit(Panomics®, Affymetrix Inc, Santa Clara, CA, USA): eotaxin, G‐CSF,fractalkine,IFN‐γ,IL‐10,IL‐1RA,IL‐1β,IL‐6,IL‐8,IP‐10,MCP‐1,MIP‐1α,MIP‐1β,TNF‐α,VEGF,ICAM‐1,VCAM‐1andRANTES(seeSupplementarymaterials).UrineMCP‐1wasalso analysed (R&D®Systems,Minneapolis,MN,USA).CytokinesIL‐10,IL‐1RAandIL‐1β were excluded from statistical analyses because their levelswereoutofthedetectionlimitinmorethan30%ofthesamples.
Comparisons of normally distributed continuous variables weremadewithStudent'st testorANOVA.Comparisonsofnon‐normallydistributed continuous variables were made with Mann‐WhitneyUorKruskal‐Wallis tests.Results for continuousvariables areex‐pressed asmedian and interquartile range (IQR). Categorical vari‐ableswereexpressedasnumberandpercentageandcomparedwiththechi‐squaretestorFisherexacttest.Survivalcurveswerecalcu‐latedwithKaplan andMeiermethodand comparedwith log‐ranktest.Duetothelackofpriorknowledgeontherelevanceofcut‐offlevelsforquantitativevariables,wedecidedtousetheunsupervised
criterionofthemedianvalue.MultivariateCoxregressionwasper‐formedtoidentifytheindependentfactorsassociatedwithmortal‐ity.Therewasnospecificcalculationofthesamplesize.However,basedonpreviousexperiencestudiesinpatientswithcirrhosis,14,18 itwasconsideredthatmorethan100patientshadtobeincludedinthisstudytoachieveasignificantnumberofoutcomesintermsoflackofAKIresolutionand3‐monthmortality.Allstatisticalanalyseswere performed using SPSS statistical package, version 23.0. Thesignificancelevelforalltestswassetat0.05two‐tailed.
2.5 | Systems biology analysis
We used a systems biology approach based on artificial neuralnetworks supervised algorithm (ANN algorithm) that measuresthestrengthofrelationshipsbetweengroupsofhumanproteins.19 Measuresarebasedonthetopologyofthenetwork,andthetrainingset isderivedmostlyfromdrugeffects.Theconditionsusedwerethose defined in BED (Biological Effectors Database, AnaxomicsBiotech, Barcelona, Spain) a hand‐curated collection of scientificknowledgerelatingbiologicalprocessestotheirmoleculareffectors.TheANNalgorithmprovidesapredictivescore(from0%to100%)that quantifies the amount and strengthof relationships betweentheevaluatedproteins.EachscoreisassociatedwithaP valuethatdescribes the probability of the results being a true positive re‐sult19,20(seeSupplementarymaterials).
3 | RESULTS
3.1 | Baseline characteristics of patients
Thebaselinecharacteristicsofpatientswithdecompensatedcirrho‐sisincludedinthisstudycategorizedinto3groups(noAKI,hypovol‐aemia‐inducedAKIandHRS‐AKI)areshowninTable1.Ofinterest,bacterial infectionsweremore common in patientswithHRS‐AKIcompared to the other two groups, a finding consistent with theknownroleofbacterialinfectionsastriggeringfactorsofHRS‐AKI.Asexpected,thefrequencyandseverityofACLFwerehigherinpa‐tientswithHRS‐AKIcomparedtothatofpatientswithhypovolae‐mia‐inducedAKI.
3.2 | Systemic inflammatory response and cytokine levels
TheprevalenceofSIRS,leucocytecountandserumCRPlevelswerehigherinpatientswithHRS‐AKIcomparedtotheothertwogroups(Table2).Withrespecttocytokinelevels,patientswithHRS‐AKIhadacytokineprofiledifferentfromthatintheothertwogroups,withstatisticallysignificantdifferencesinthelevelsofseveralcytokines,including higher urinary levels ofMCP‐1 and plasma IL‐6, TNF‐α, VCAM‐1,andIL‐8,andlowerlevelsofMIP1‐αandfractalkine.
TofurtherexplorethecytokineprofileofHRS‐AKI,weassessedwhethertheincreasedcytokinelevelsfoundinthesepatientscouldbe related to concomitant bacterial infections that were more
4 | SOLÉ et aL.
commoninthisgroupofsubjects.Withthisobjective,wecomparedcytokine levels inpatientswithHRS‐AKIcategorizedaccording topresenceorabsenceofinfection(42and16patients,respectively)(SupplementaryTable1).Outofthe5cytokinesthatweredifferen‐tiallyincreasedinpatientswithHRS‐AKI,onlyplasmaIL‐6wassig‐nificantlyhigherinpatientswithHRS‐AKIassociatedwithinfectionscomparedtothosewithoutinfections(59(21‐180)vs23(12‐55)pg/mL,respectively,P=0.02).However,itispossiblethatafurtherag‐gravationof the increased systemic inflammation duringHRS‐AKIassociatedwithbacterialinfectionscouldnotbedetectedduetothelownumberofpatientswithHRS‐AKIwithoutinfections.
Wealso investigatedwhetherreversalofkidney impairment inpatientswithHRS‐AKIwasassociatedwithchangesincytokinepro‐file.Tothispurpose,wecomparedcytokinelevelsbeforeandafterkidneyfunctionimprovementin24patientswithtype‐1HRStreatedwithvasoconstrictors andalbumin.Asexpected, serumcreatininedecreased significantly after treatment (from 2.9 to 1.3mg/dL;
P<0.001).Outofall cytokinesevaluated,onlyplasmaTNF‐α and RANTESdecreasedsignificantlyandMIP‐1αincreasedwithreversalofkidneydysfunction(Table3).
SincemanypatientswithHRS‐AKImeetthecriteriaofACLF,weinvestigatedwhethertheincreasedlevelsofinflammatorycytokinesfound inHRS‐AKIwerepotentially related to thepresenceofcon‐comitantACLF.To thisaim,wecategorizedpatientswithHRS‐AKIaccordingtothepresenceorabsenceofACLF.AsshowninTable4,patientswithHRS‐AKIwithassociatedACLFhadlevelsofcytokinesthatweresimilar to thoseofpatientswithHRS‐AKIwithoutACLF.Moreover,thelevelsofcytokinesdidnotcorrelatewithACLFsever‐ity,exceptforhigherlevelsofIL‐8andICAM‐1inpatientswithACLFgrades2‐3vsthoseofpatientswithgrade1(Table5).Tofurtherex‐ploretherelationshipbetweenAKIandACLF,wecomparedinflam‐matorycytokinelevelsinpatientswithACLFcategorizedaccordingtoAKItype,eitherHRS‐AKIorhypovolaemia‐inducedAKI.PatientswithACLFassociatedwithHRS‐AKIhadhigherlevelsofIL‐6,TNF‐α andurinaryMCP‐1andlowerlevelsoffractalkineandMIP1‐α com‐paredtothehypovolaemia‐induceAKIcounterparts(datanotshown).
3.3 | Relationship of systemic inflammatory response and cytokine levels with kidney outcome and patient survival
patientshadpersistentAKI.PatientswithpersistentAKIhadsignifi‐cantlyhigherbaseline leucocytecountandCRPlevelsandplasmaIL‐6andVCAM‐1compared to thoseofpatientswithAKI resolu‐tion. InthegroupofpatientswithHRS‐AKI,thosewithpersistentAKI(27patients,47%)showedsignificantlyhigherleucocytecountandplasmalevelsofIP‐10andVCAM‐1comparedtothosewhohadresolutionofHRS‐AKI(31patients,53%),yetdifferencesoftheselattertwocytokineswerebarelysignificant(Table6).
At the end of the 3‐month follow‐up period, 110 patients(69%)werealive,34(21%)haddied,and15(9%)hadbeentrans‐plantedand2were lost tofollow‐up. Interestingly,anumberofinflammatoryparametersandcytokinescorrelatedwith3‐monthmortality: leucocyte count, CRP levels and plasma IL‐6, IL‐8,TNF‐α, ICAM‐1 andVCAM‐1. Inmultivariate analysis, includingthoseinflammatoryvariablesthatwereassociatedwithmortalityintheunivariateanalysis,onlyplasmaVCAM‐1wasanindepen‐dentpredictivefactorof3‐monthmortality.PlasmaVCAM‐1wasalso the only inflammatory marker independently predictive of3‐monthmortality inthegroupofpatientswithHRS‐AKI (3156(2029‐4349)vs1944 (1535‐2829)ng/mL, forpatientswhodiedandsurvivedat3months,respectively;P<0.01),evenwhenad‐justedforthepresenceandseverityofACLF.Survivalprobabilityin patientswithHRS‐AKI according toVCAM‐1 levels is shownin Figure 1.
3.4 | Comparison of cytokine profile in HRS‐AKI with other chronic conditions using with systems biology approach
We used a systems biology approach based on artificial neuralnetworks that measures the strength of relationships betweengroups of human proteins in order to compare the extent of sys‐temic inflammationinHRS‐AKIwiththatofotherconditionschar‐acterizedbywell‐definedandmarkedsystemicinflammatorystatus.Interestingly,thecytokinepatternofHRS‐AKIwasrelatedtothatofanumberofinflammatoryconditions,includingcysticfibrosis,rheu‐matoidarthritis,systemiclupuserythematosus,Crohn'sdiseaseandulcerativecolitis(predictivevalue>70)(Figure2).
4 | DISCUSSION
TheresultsofthecurrentstudyshowthatpatientswithHRS‐AKIhave marked systemic inflammation with altered cytokine pro‐file compared to that of patients with decompensated cirrhosiswithoutAKIand,most interestingly, topatientswithAKIduetohypovolaemia. The systemic inflammatory response in HRS‐AKIdoesnot seem tobe related topresenceofbacterial infections,concomitantACLF or intensity of kidney dysfunction and is not
TA B L E 2 Comparisonofsystemicinflammatorymarkersandplasmaandurinecytokinelevelsbetweenthe3groupsofpatients
No AKI (N = 44)Hypovolaemia‐induced AKI (N = 63) HRS‐AKI (N = 58) P value
normalized by improvement of kidney function with pharmaco‐logical therapy. Interestingly, the intensity of the inflammatoryresponseiscorrelatedwithkidneyandpatientoutcomesinsuchawaythatincreasedlevelsofsomeinflammatorymarkers,particu‐larlyVCAM‐1, are associatedwith lack of resolution ofAKI andmortality.
In the current study, a large number of consecutive patientswithcirrhosisandHRS‐AKIwereinvestigatedforthepresenceofsystemic inflammatory response as assessed by a large numberof inflammatoryandanti‐inflammatorycytokinesusingmultiplextechnology.Acontrolgroupofpatientswithdecompensatedcir‐rhosiswithoutAKIwas included forcomparison.Agroupofpa‐tientswithdecompensatedcirrhosiswithAKIduetohypovolaemia
wasalsostudied.ThistypeofAKIwasselectedascomparatorforHRS‐AKIbecauseinbothconditionsAKIisofprerenalorigin,yetthe underlying pathogenic cause is very different.While a con‐tractedbloodvolume is thecauseof renalhypoperfusion in theformer,the impairmentofkidneyfunctioninthe latter isrelatedtooppositecirculatoryfeatures,namelymarkedlydilatedvascularbed,particularlyinthesplanchniccirculation.2‐4Theresultsofthecurrentstudyclearlyshowthatasdecompensatedcirrhosispro‐gressestowardsHRS‐AKI,thereisprogressiveincreaseininflam‐matorystatuswithsignificantlyincreasedlevelsofsomepowerfulinflammatorycytokines.Previousstudieshaveshownthatplasmalevels of inflammatory cytokines are significantly increased indecompensated compared to compensated cirrhosis, suggesting
TA B L E 3 Systemicinflammatorymarkersandplasmaandurinecytokinelevelsinpatientswithtype1HRS‐AKIbeforeandaftereffectivetreatmentwithterlipressinandalbumin
Baseline (N = 24)Terlipressin and Albumin (N = 24) P value
the existence of an inflammatory driving force that occurswiththe progression of the disease.8,9 Whether this inflammatorydrivingforceiscauseorconsequenceofprogressionofliverdis‐easeisnotknown.Ourdataconfirmthatthisinflammatorystatusacrossdecompensated cirrhosis increases evenmore as thedis‐easeprogressestowardsHRS‐AKIwhichisconsideredoneofthelatest stages of cirrhosis, given its highmortality rate.Our datathereforeareinagreementwiththerecentlyproposedtheoryof
systemic inflammation driving the complications of cirrhosis.15 Further support to this theory comes fromour systems biologyanalysesshowingthatsystemicinflammationincirrhosisissimilartothatfoundinsomekeychronic inflammatoryconditions,suchasinflammatoryboweldiseases,rheumatoidarthritisorsystemiclupuserythematosus.
TA B L E 5 ComparisonofsystemicinflammatorymarkersandplasmaandurinecytokinelevelsinpatientswithHRS‐AKIassociatedwithACLFclassifiedaccordingtoACLFseverity
thattheincreasedinflammatorystateisnotrelatedtoACLF.Firstly,patientswithHRS‐AKIbutwithoutALCFhadplasmacytokinelev‐elsthatwerenotsignificantlydifferentfromthosefoundinpatientswith HRS‐AKI with ACLF.Moreover, cytokine levels were largelyunrelatedtoACLFgrade.Ontheotherhand,cytokineprofileofpa‐tientswithHRS‐AKIwasnoticeablydifferentfromthatofpatientswithACLF associatedwith hypovolaemia‐inducedAKI, suggestingthatcytokineprofilewasmostlyrelatedto‘hepatorenalsyndrome’andnottoACLF.Nevertheless,thesefindingsshouldbetakenwithcautionbecauseof the relatively lownumberofpatients included
whichpreventedperformingapropensityscorematchinganalysis.Furtherstudiesareneededtotrytodissectoutwhethersystemicinflammation is due to hepatorenal syndrome ‘per se’ or toACLF,orboth.
A final issue thatdeservesdiscussion is thatpatients inwhomHRS‐AKIpersistedshowedhigherlevelsofsomeinflammatorymark‐ers,themostimportantofwhichappearstobeVCAM‐1.Moreover,VCAM‐1wasalsoanindependentpredictivefactorofsurvivalinthewholeseriesofpatients.VCAM‐1isaninflammatorymediatorthatplaysacentralroleintriggeringtheprocessofsystemicinflamma‐tion in responsetoseveral stimulibyhelping recruit inflammatorycellsoutsideofthesystemiccirculation.21Fewpreviousstudieshaveshown thepotential relevanceofVCAM‐1asprognostic indicatorofpatientswithcirrhosis.22Ourresultsextendtheseobservationsby showing that among a large number of inflammatorymarkers,VCAM‐1 plasma levels are associated with lack of resolution ofHRS‐AKIandpoorsurvival.Theseresultstogetherwithfindingsofasimilarinflammatoryprofilecomparedtothatofsomeinflammatorydiseasesshed lightonthepotential roleofVCAM‐1andTNF‐α as therapeutictargetsinpatientswithadvancedcirrhosis.23
F I G U R E 1 Probabilityof3‐monthsurvivalinpatientswithHRS‐AKIcategorizedaccordingtomedianlevelsofVCAM‐1
VCAM-1< 2372 ng/ml
VCAM-1 ≥ 2372 ng/ml ------
p = 0.023
Days
Surv
ival
F I G U R E 2 Relationshipbetweensystemicinflammatorypatterninpatientsincludedinthisstudyanddifferentpathologicalconditionsbasedonanetworkanalysis.Linkwidthanddistancetothecentralpointisproportionaltothepredictivescoreofthenetworkanalysisthatquantifiesthenetworkrelationshipsbetweentheevaluatedproteins.OnlymedicallyrelevantconditionswithapredictivescorecorrespondingtoP≤0.1aredisplayed.ConditionsasdefinedinBED(BiologicalEffectorsDatabase,AnaxomicsBiotech)
| 9SOLÉ et aL.
thisalternativestrategywasnotpossible.Secondly,giventherela‐tivesmallsizesofourstudypopulationsandtheexploratorynatureofthisstudy,wedecidednottoimplementmultiplicityadjustmentstrategies.ThisstudywillrequirefuturereplicationandthefindingsingeneralandtheP valuesinparticularshouldbeinterpretedwithcaution.Thirdly,although thenumberofpatientsstudiedmayap‐pearrelativelylow,itisaquitelargesampleofpatientsconsideringthedifficultytoperformclinicalstudiesindecompensatedcirrhosisand the sample sizeofpreviouspathogenic and therapeutic stud‐ies inpatientswithHRS‐AKI.Finally, levelsofcytokines inplasmamaynotreflecttheactualconcentrationsofthesamecytokinesintissues.However,thisisalimitationthatcanbarelybeaddressedinhumanstudies.
In conclusion, patientswithHRS‐AKI havemarked increase insystemicinflammatoryprofilecomparedtothatofpatientswithoutAKIandhypovolaemia‐inducedAKI,whichappearstobeindepen‐dentofassociatedACLF,andsimilartoinflammationobservedinkeysystemicinflammatorydiseases,suchaslupuserythematosusorin‐flammatoryboweldisease.Interestingly, inpatientswithHRS‐AKI,lackofAKIresolutionandsurvival is linkedtosomespecificcyto‐kines,particularly,VCAM‐1.
ACKNOWLEDGEMENTS
We thankNicki VanBerckel for the administrative support.Wealso thank the faculty andnursesof the LiverUnit, and thepa‐tients who participated in this study and their families.We areindebtedtotheBiobankcorefacilityoftheIDIBAPSforthetech‐nicalhelpprovided.
CONFLICT OF INTEREST
PG reports Investigator Research grant andAdvisory BoardworkfromGrifols,InvestigatorResearchgrantandAdvisoryBoardfromGilead, Investigator Research grant from Mallinckrodt, AdvisoryBoardforPromethera,AdvisoryBoardforMartin‐Pharmaceuticals,grants from Ferring‐Pharmaceuticals, grants and Advisory BoardWorkfromSequana,outsidethesubmittedwork.Nootherauthorshaveanydeclaredinterests.
AUTHOR CONTRIBUTIONS
Theauthorshaveallcontributedtothismanuscriptandapprovetheversionofthissubmission.CScontributedtotheconceptionandde‐signofthisstudy,acquisitionofdata,theanalysisandinterpretationof the data and drafting themanuscript; PH,MC, RM,UC, JMM,IG,EP,LN,GDP,AJ,NF,MMR,JF,WJ,participatedintheanalysesoftheresults, interpretationofdata,and/orcriticalrevisionofthemanuscript.FTcontributedtostatisticalanalysisandinterpretationofdata.PGandESparticipatedinthisstudyconcept,interpretationof thedata,drafting themanuscript,critical revisionof themanu‐scriptforimportantintellectualcontent,obtainedfundingandstudysupervision.
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SUPPORTING INFORMATION
Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.
How to cite this article:SoléC,SolàE,HuelinP,etal.Characterizationofinflammatoryresponseinhepatorenalsyndrome:Relationshipwithkidneyoutcomeandsurvival.Liver Int. 2019;00:1–10. https://doi.org/10.1111/liv.14037
A part dels dos articles originals publicats que formen part d’aquesta Tesi
Doctoral, durant aquest període he participat també en les següents
publicacions relacionades amb aquesta àrea d’investigació:
- Solà E*, Solé C*, Simón-Talero M, Martín-Llahí M, Castellote J, Garcia-Martínez R, Moreira R, Torrens M, Márquez F, Fabrellas N, de Prada G, Huelin P, Lopez Benaiges E, Ventura M, Manríquez M, Nazar A, Ariza X, Suñé P, Graupera I, Pose E, Colmenero J, Pavesi M, Guevara M, Navasa M, Xiol X, Córdoba J, Vargas V, Ginès P. Midodrine and albumin for prevention of complications in patients with cirrhosis awaiting liver transplantation. A randomized placebo-controlled trial. J Hepatol. 2018 Dec;69(6):1250-1259. doi: 10.1016/j.jhep.2018.08.006. *co-autores.
Articles originals
- Piano S, Schmidt HH, Ariza X, Amoros A, Romano A, Hüsing-Kabar A, Solà E, Gerbes A, Bernardi M, Alessandria C, Scheiner B, Tonon M, Maschmeier M, Solé C, Trebicka J, Gustot T, Nevens F, Arroyo V, Gines P, Angeli P; EASL CLIF Consortium. Association Between Grade of Acute on Chronic Liver Failure and Response to Terlipressin and Albumin in Patients With Hepatorenal Syndrome. Clin Gastroenterol Hepatol. 2018 Jan 31. pii: S1542-3565(18)30105-8. doi: 10.1016/j.cgh.2018.01.035.
- Graupera I, Coll M, Pose E, Elia C, Piano S, Solà E, Blaya D, Huelin P, Solé C, Moreira R, de Prada G, Fabrellas N, Juanola A, Morales-Ruiz M, Sancho-Bru P, Villanueva C, Ginès P. Adipocyte Fatty-Acid Binding Protein is Overexpressed in Cirrhosis and Correlates with Clinical Outcomes. Sci Rep. 2017 May 12;7(1):1829. doi: 10.1038/s41598-017-01709-0.
- Huelin P, Piano S, Solà E, Stanco M, Solé C, Moreira R, Pose E, Fasolato S, Fabrellas N, de Prada G, Pilutti C, Graupera I, Ariza X, Romano A, Elia C, Cárdenas A, Fernández J, Angeli P, Ginès P. Validation of a Staging System for Acute Kidney Injury in Patients With Cirrhosis and Association With Acute on Chronic Liver Failure. Clin Gastroenterol Hepatol. 2016 Oct 5. pii: S1542-3565(16)30869-2. doi: 10.1016/j.cgh.2016.09.156.
- Ariza X, Graupera I, Coll M, Solà E, Barreto R, García E, Moreira R, Elia C, Morales-Ruiz M, Llopis M, Huelin P, Solé C, Fabrellas N, Weiss E, Nevens F, Gerbes A, Trebicka J, Saliba F, Fondevila C, Hernández-Gea
79
V, Fernández J, Bernardi M, Arroyo V, Jiménez W, Deulofeu C, Pavesi M, Angeli P, Jalan R, Moreau R, Sancho-Bru P, Ginès P; CANONIC nvestigators, EASL CLIF Consortium. Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis. J Hepatol. 2016 Jul;65(1):57-65.doi: 10.1016/j.jhep.2016.03.002
- Isabel Graupera, Elsa Solà, Núria Fabrellas, Rebeca Moreira, Cristina Solé, Patricia Huelin, Gloria de la Prada, Elisa Pose, Xavier Ariza, Alessandro Risso, Sonia Albertos, Manuel Morales-Ruiz, Wladimiro Jiménez, Pere Ginès. Urine Monocyte Chemoattractant Protein-1 Is an Independent Predictive Factor of Hospital Readmission and Survival in Cirrhosis. PLoS One. 2016 Jun 30;11(6):e0157371. doi: 10.1371/journal.pone.0157371.
- Solé C and Solà E. Uptade on acute-on-chronic liver failue. Gastroenterol Hepatol. June 2017. doi:10.1016/j.gastrohep.2017.05.012
Revisions
- Solé C, Pose E, Solà E, Ginès P. Hepatorenal syndrome in the era of Acute Kidney injury. Liver Int. 2018 May 30. doi: 10.1111/liv.13893.
- Cristina Solé, Elsa Solà and Pere Ginès. Acute on chronic liver failure: The syndrome. En: Vicente Arroyo and Mauro Bernardi. 1rst International Meeting. Systemic Inflammation and Organ Failure in Cirrhosis. The Acute-on-Chronic liver Failure Syndrome (ACLF). Spain: Elsevier; 2016. P. 11-14.
Capítols de llibre
- Cristina Solé, Andrés Cardenas and Pere Ginès. 2019. “Chapter 169. The patient with hepatorenal syndrome. In: Oxford Textbook of Clinical Nephrology. Edited by Neil N. Turner, Norbert Lameire, et al. 5th edition (Submitted).
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8.2 Comunicacions a congressos
Els projectes que formen aquesta Tesi Doctoral han generat les següents
comunicacions a congressos:
- Cristina Solé; Elsa Solà; Manuel Morales-Ruiz, Guerau Fernàndez;
Patricia Huelin; Isabel Graupera, et al. Caracterització de la resposta
inflamatòria de la síndrome de “Acute-on-chronic liver failure” (ACLF) i
relació amb el pronòstic. Categoria: pòster. XXV Congrés de la Societat
Catalana de Digestologia. Reus, Gener 2016.
Congressos nacionals:
- Cristina Solé; Elsa Solà; Manuel Morales-Ruiz, Guerau Fernàndez;
Patricia Huelin; Isabel Graupera, et al. Caracteritzación de la respuesta
inflamatoria del síndrome de “Acute-on-chronic liver failure” (ACLF) i
relación con el pronóstico. Categoria: poster. 41º Congreso (AEEH).
Madrid. Febrer 2016.
- Cristina Solé, E.Solà, M.Morales, et al. Caracterització de la resposta
inflamatòria sistèmica de la insuficiència renal aguda-síndrome
hepatorenal (AKI-SHR) a la cirrosi. Paper rellevant de la il-6, TNF-a i
VCAM. Categoria: pòster. XXVII Congrés de la Societat Catalana de
Digestologia. Lleida, Gener 2018
- Cristina Solé, E.Solà, M.Morales, et al. Caracterización de la respuesta
inflamatoria sistémica en la insuficiencia renal aguda-síndrome
hepatorenal (AKI-SHR) en la cirrosis. Papel relevante de la IL-6, TNF-Α y
VCAM.Categoria: poster. 43 Congreso Anual de la Asociación
Española para el Estudio del hígado (AEEH). Febrer 2018.
- Cristina Solé, Elsa Solà, Manuel Morales-Ruiz, Guerau Fernàndez,
Patricia Huelin, Isabel Graupera, Rebeca Moreira, Gloria De Prada,