... Fanali C, Cabras T, Castagnola M. Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us? J Sep Sci 2008;. 31(11):1948-63.
DOCTORAL THESIS
OSAMA MOSA
Ljubljana, 2016
I
OSAMA MOSA
Vloga proteinov Klotho in CYR61 pri zgodnjem odkrivanju akutne poškodbe ledvic po operaciji srca. The role of Klotho and CYR61 proteins in early diagnosis of acute kidney injury after cardiac surgery
DOCTORAL THESIS
LJUBLJANA, 2016
II
Supervisor: Assoc. Prof. Dr. Milan Skitek, PhD, Eur Spec Lab Med Co-mentor: Assist. Prof. Dr. Aleš Jerin, PhD, Eur Spec Lab Med Commission: Prof. Dr. Darko Černe, PhD, Eur Spec Lab Med Prof. Dr. Janja Marc, PhD, Eur Spec Lab Med
Declaration: I declare that the submitted doctoral thesis is independent work which I have performed under supervision of Assoc. Prof.Dr. Milan Skitek, PhD, MSc Pharm. (Mentor) and Ass Prof Dr Aleš Jerin, PhD (Co-mentor). The research work presented in this study was conducted at Institute of Clinical Chemistry and Biochemistry, The University Medical Center Ljubljana, Slovenia. OSAMA MOSA
Izjava: Spodaj podpisani Osama Mosa, M.Sc. izjavljam, da sem doktorsko disertacijo izdelal samostojno pod mentorstvom izr. prof. dr. Milana Skitka, (mentor) in doc. dr Aleša Jerina, (somentor). Raziskave, predstavljene v tem doktorskem delu, so bile izvedene na Kliničnem inštitutu za klinično kemijo in biokemijo, Univerzitetni klinični center Ljubljana (UKCL), Slovenija. OSAMA MOSA
III
Firstly, this thesis is dedicated to my “Father”, “Mother”, “Wife”, “Kids” and “Sisters” Who believe in me, supporting me in all my life, lived with me the hours of anxiety and happiness and never suspect in my abilities or try to break my hopes. To my dearest Asmaa Fahmy, may this thesis reminds you that our initial dreaming is the first step towards success. As well, I have never forgotten my mentors for them continuously supports, struggling to finish this work in the acceptable aspects and never let me down along my study.
IV
“The great successful men of the world have used their imagination? They think ahead and create their mental picture in all its details, filling in here, adding a little there, altering this a bit and that a bit, but steadily building - steadily building”.
Robert Collier
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Doctoral Thesis
ABSTRACT
ABSTRACT Acute kidney injury (AKI) is a critical problem accompanied with cardiac surgery as one of the adverse complications that contribute to prolonged hospitalization period and/or increase morbidity and mortality rates, surpassing 40% among cardiac ICU patients. Cardiac surgery with cardiopulmonary bypass (CPB) reproduces globally every year leading to volume depletion as a result of cardiac output exhaustion and stimulation of renin-angiotensin system with renal vasoconstriction that increases the extent of AKI among hospitalized patients. It is well known that AKI may worsen kidney function by time depending on slowly traditional biomarker “serum creatinine” which tardily increases after 2 days of renal tubular injury indicating that 50% of kidney function is lost and creatinine kinetics is influenced by ethnicity, gender, age, dietary protein intake, muscle mass and medications that didn’t reflect precisely the prognosis of AKI over time.
Therefore, the needs for novel biomarkers are important situations to improve sensitivity, specificity of clinical diagnosis and potentiate the efficiency of treatment to prevent irreversible consequences. In the present doctoral work, the following hypotheses will be tested: 1)
Plasma/serum or urinary levels of Klotho and CYR61 are early prognostic biomarkers of AKI after cardiac surgery.
2)
Klotho and CYR61 are markers of renal tubular dysfunction at the different levels of AKI after cardiac surgery.
3)
The combinations of Klotho and CYR61 with other markers (f.e. NGAL/Klotho or KIM-1/Klotho ratios) will increase the prognostic efficiency of AKI after cardiac surgery.
Our study covered 50 patients admitted to cardiac surgery department in the University Medical Center Ljubljana and fulfilled the inclusion criteria of selection. The classification of patients into AKI group and non-AKI group relies on KDIGO (Kidney Disease Improving Global Outcomes) that declared its validation for AKI staging and severity in the recent ADAQI consensus, retained almost advantages of both RIFLE and AKIN criteria in a harmonized manner. Serum levels of creatinine, cystatin C, CYR61 and klotho were measured at five time intervals before and within 48 hours of CPB. Adding cystatin C levels to creatinine was thought to be beneficial from various aspects. Firstly cystatin C with creatinine levels were important in GFR estimation by MDRD equation as GFR
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Doctoral Thesis
ABSTRACT
when decline in proximal renal tubules, cystatin C levels arises at renal, urinary and peripheral blood levels. Secondly, the measurements of cystatin C levels preoperatively and after one day of cardiothoracic surgery, were mentioned to be indicative of AKI development. Within the thesis, the serum levels of creatinine and cystatin C showed shared behaviors across AKI and non-AKI groups. In the AKI group, both serum creatinine and cystatin C levels were synchronically increased than baseline values after 2 h of CPB and reached a maximal peak after the second day of CPB. While in the non-AKI group, both of them suffered from a sudden decline in the beginning and after a while showed slightly different situations where creatinine increased after 1 day and fall-down within 2 days to reach the baseline value, but cystatin C was insistently elevated after 2 days overcoming the initial baseline.
CYR61 was a mysterious protein and its role in the body is still not fully understood in spite of being involved with wound healing, fibrosis, inflammation, arthritis, vascular diseases and cancer. In renal diseases, this protein was studied experimentally at the urinary and mRNA levels but not in the blood levels and its previous results were optimistic. We have the priority to assess this protein at serum level on human subjects due to the facility to be measured by sensitive ordinary analytical techniques and less affected by other species like the urinary levels. In the thesis, serum CYR61 baseline levels were found to be high in the non-AKI group than in the AKI group and within 2 days of CPB, its levels tend to decrease in the non-AKI by half value of the starting baseline point and reciprocally rise up in the AKI group overcoming initial baseline without any statistical significance. As well, serum CYR61 levels might differentiate between stages I and II of the AKI groups with a slight significance that could suggest the effect of limited numbers of studied patients and future implications in AKI stratification and stage grading if applied on a large scale.
Eventually, working on Klotho protein was an important precedent after revealing possible interconnections in the body, including it role with bone-derived hormone (FGF-23), anti-aging character, and antagonist for oxidative stress and released in tissues normally including renal tubules. Moreover, almost every study conducted on klotho levels on animal models concluded that the evident reduction happened after ischemic renal injury at mRNA transcripts, urine and plasma levels. In the thesis, baseline levels of klotho in the AKI group are explicitly raised than in the non-AKI group, continuing upwardly to the same extent after 2h of CPB and eventually declined within 2 days of surgery.
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Doctoral Thesis
ABSTRACT
Interestingly, testing the kinetic performance of these biomarkers regarding AKI situation, indicates that klotho and cystatin C were close together overcoming CYR61. As well, the combination of klotho and creatinine increases the diagnostic utility against AKI than CYR61. Ultimately, according to our emerged results we were able to accept both first, third hypotheses and partially accept the second one.
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Doctoral Thesis
POVZETEK
POVZETEK Akutna ledvična okvara (ALO) je resen problem pri operacijah srca kot eden od zapletov, ki podaljšajo hospitalizacijo in/ali zvečajo obolevnost in smrtnost, ki pri srčnih bolnikih v enotah intenzivne nege (EIN) presegata 40 %. Operaciji srca s kardiopulmonalnim obvodom (KPO) prispevajo k zmanjšanju volumna, skupaj s stimulacijo sistema renin-angiotenzin in renalno vazokonstrikcijo zvečajo obseg ALO pri hospitaliziranih bolnikih. Znano je, da lahko ALO posledično poslabša ledvično funkcijo. Uveljavljen biooznačevalec serumski kreatinin se zviša šele po dveh dneh od tubulne okvare, ko ledvična funkcija upade za 50 %. Na kinetiko tega označevalca močno vpliva etnična pripadnost, spol, starost, vnos beljakovin, mišična masa in zdravila. Da bi preprečili ireverzibilne posledice, so potrebni novi biooznačevalci, ki bi izboljšali občutljivosti in specifičnosti klinične diagnoze in zvečali učinkovitost zdravljenja. V tej doktorski dela bodo testirali naslednje hipoteze: 1) Plazma / serum ali ravni sečil z Klotho in CYR61 so zgodnji napovedni biomarkerjev ALO po operaciji srca. 2) Klotho in CYR61 so označevalcev ledvično tubularno disfunkcijo na različnih ravneh ALO po operaciji srca. 3) kombinacije Klotho in CYR61 z drugimi označevalci (npr. NGAL / Klotho ali KIM-1 razmerja / Klotho) se bo povečala napovedni učinkovitost ALO po operaciji srca. Naša raziskava je vključevala 50 bolnikov z načrtovano operacijo srca, ki so bili sprejeti na oddelek srčne kirurgije v Univerzitetnem kliničnem centru Ljubljana in so izpolnjevali vključitvene kriterije. Bolniki so bili razvrščeni v ALO in brez ALO skupino glede na kriterij KDIGO (Kidney Disease Improving Global Outcomes), ki je bil potrjen za oceno stopnje ALO z nedavnim ADAQI konsenzom in združuje večino prednosti RIFLE in AKIN kriterijev. Merili smo serumski nivo kreatinina, cistatina C, CYR61 in Klotho pri petih časovnih intervalih pred operacijo in 48 ur po CPB. Meritev cistatina C skupaj s kreatininom naj bi bila koristna iz več vidikov. Nivoja cistatina C in kreatinina sta pomembna pri oceni GFR z MDRD formulo, ob upadu GFR nivo cistatina C naraste ledvicah, urinu in periferni krvi. Poleg tega naj bi bil nivo cistatina C pred operacijo in en dan po operaciji pokazatelj razvoja ALO. V naši raziskavi sta se serumska nivoja kreatinina
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Doctoral Thesis
POVZETEK
in cistatina C podobno spreminjala v ALO in brez ALO skupini. V ALO skupini sta bila oba zvišana nad začetno vrednost dve uri po CPB in sta dosegla najvišjo vrednost drugi dan po CPB. V brez ALO skupini sta se oba nivoja v začetku hitro znižala, zatem pa se je potek razlikoval: kreatinin se je zvišal po enem dnevu in po dveh dneh upadel na izhodiščno vrednost, cistatin C pa je ostal zvišan nad izhodiščno vrednostjo tudi po dveh dneh. Vloga CYR61 v telesu še ni popolnoma poznana. Sodeluje pri celjenju ran, fibrozi, vnetju, artritisu, žilnih boleznih in raku. Pri boleznih ledvic so preučevanja nivoja v urinu in meritev nivoja mRNA dala spodbudne rezultate, nivoja v krvi pa še niso preučevali. V raziskavi smo v tem kontekstu prvi preučevali serumski nivo humanega CYR61. Višje začetne vrednosti smo izmerili v brez ALO skupini. Po dveh dneh po CPB so vrednosti v brez ALO upadle na polovico začetne vrednosti, medtem ko so bile v ALO skupini rahlo - vendar ne statistično značilno višje od izhodiščnih. Nivo CYR61 bi lahko bil uporaben za razlikovanje stopenj I in II v ALO skupini, a ta povezava je zaradi omejenega števila bolnikov v naši raziskavi dosegla le mejno statistično značilnost.
Klotho ima v telesu razne vloge: pri procesih staranja, kot antagonist oksidativnega stresa in delovanje skupaj s hormoni, kot je FGF-23. Sprošča se v raznih tkivih, tudi v ledvičnih tubulih. Z raziskavami na živalih so ugotovili, da se po ishemični poškodbi ledvic zmanjša nastajanje mRNA in tudi nivo Klotho v urinu in plazmi. V naši raziskavi je bil izhodiščni nivo Klotho višji v ALO kot v brez ALO skupini, razlika med skupinama je bila podobna tudi dve uri po CPB. Vrednosti so se znižale dva dni po operaciji. Gibanje nivoja merjenih biooznačevalcev kaže, da imata Klotho in cistatin C pri ALO večji potencial kot CYR61. Kombinacija Klotho in kreatinina pa bi lahko še zvečala diagnostično uporabnost pri ALO. Na koncu smo glede na naše rezultate lahko potrdili prvo in tretjo hipotezo ter delno tudi drugo.
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Doctoral Thesis
TABLE OF CONTENTS
TABLE OF CONTENTS ABSTRACT ............................................................................................................................. 1 POVZETEK ............................................................................................................................ 4 LIST OF ABBREVIATIONS ................................................................................................. 8 INTRODUCTION CHAPTER ............................................................................................ 10 UVODNO POGLAVJE ......................................................................................................... 16 AIM AND DESCRIPTION OT THE DOCTORAL THESIS CHAPTER ...................... 17 NAMEN IN OPIS DOKTORSKE DISERTACIJE ............................................................ 19 MATERIALS AND METHODS CHAPTER...................................................................... 21 ARTICLES CHAPTER......................................................................................................... 23 CHAPTER 1/POGLAVJE1: FIRST SCIENTIFIC PAPER--------------------------------------23 EVALUATION OF SERUM CYSTEINE-RICH PROTEIN 61 AND CYSTATIN C LEVELS FOR ASSESSMENT OF ACUTE KIDNEY INJURY AFTER CARDIAC SURGERY.
CHAPTER 2/POGLAVJE2: SECOND SCIENTIFC PAPER-----------------------------------40 SERUM KLOTHO AS A MARKER FOR EARLY DIAGNOSIS OF ACUTE KIDNEY INJURY AFTER CARDIAC SURGERY.
CHAPTER 3/POGLAVJE3: REVIEW ARTICLE------------------------------------------------54 VALIDITY OF KLOTHO, CYR61, AND YKL-40 AS IDEAL PREDICTIVE BIOMARKERS FOR ACUTE KIDNEY INJURY.
RESULTS CHAPTER ........................................................................................................... 75 GENERAL DISCUSSION CHAPTER ................................................................................ 90 CONCLUSION CHAPTER .................................................................................................. 98 SKLEPNO POGLAVJE ........................................................................................................ 99
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Doctoral Thesis
TABLE OF CONTENTS
REFRENCES ....................................................................................................................... 100 AUTHOR’S BIOGRAPHY (CURRICULUM VITAE) .................................................. 110 AUTHOR’S BIBLIOGRAPHY .......................................................................................... 115 ACKNOWLEDGEMENTS ................................................................................................ 116
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Doctoral Thesis
LIST OF ABBREVIATIONS
LIST OF ABBREVIATIONS ADQI
Acute Dialysis Quality Initiative.
AKI
Acute Kidney Injury.
AKIN
Acute Kidney Injury Network.
AUC
Area under the curve.
BMI
Body Mass Index.
BUN
Blood Urea Nitrogen.
CKD
Chronic kidney Disease.
CPB
Cardiopulmonary bypass.
CSA-AKI
Cardiac surgery associated AKI.
CT
C-terminal Domain.
CVD
Cardiovascular disease.
CYR61
Cysteine Rich Protein 61.
EBM
Evidence- based Medicine.
EDNO
Endothelial-derived Nitric oxide.
eGFR
Estimated glomerular filtration rate.
ESRD
End-stage Renal Disease.
FET
Fisher's exact test.
FGF-23
Fibroblast growth factor-23.
FOXOs
Forkhead box proteins.
HSP-70
Heat shock protein-70.
ICU
Intensive Care Unit.
IGFBR
Insulin-like growth factor binding protein.
IL-18
Interleukin-18.
IRI
Ischemic Reperfusion injury.
KDIGO
Kidney Disease improving Global Outcomes.
MCP-1
Monocyte chemoattractant protein-1.
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Doctoral Thesis
LIST OF ABBREVIATIONS
MDRD
Modification of Diet in Renal Disease.
NF-κ B
Nuclear factor κB.
NKCC
Na-K-2Cl cotransporter.
PGI2
Prostacyclin.
RA
Rheumatoid Arthritis.
RAAS
Renin-angiotensin aldosterone system.
RIFLE
Risk Injury Failure Loss of Function End-stage.
ROC
Receiver operating curve.
ROS
Reactive oxygen species.
RRT
Renal Replacement Therapy.
SLE
Systemic Lupus Erythematosus.
SN-GFR
Single nephron-GFR.
SNPs
Single nucleotide polymorphisms.
SOD2
Super oxide dismutase-2.
TGF
Tubuloglomerular feedback.
TGF-β
Tumor growth factor-β.
TNF-α
Tumor necrosis factor α.
TSR
thrombospondin type 1 repeat domain.
UO
Urinary Output.
vWC
von Willebrand type C repeats domain.
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Doctoral Thesis
INTRODUCTION CHAPTER
INTRODUCTION CHAPTER Acute Kidney Injury (AKI) is a critical epidemic transition state that comes after cardiac surgery as one of the possible etiologies and characterized by rapidly progressive complications with renal function degeneration dependent on the elevation of serum creatinine levels compared with reference baseline and abrupt decline in urine output [1, 2]. Cardiac surgery is virtually multiplying annually in all over the world and usually be associated with cardiopulmonary bypass (CPB) that leads to diminished cardiac output, activation of the reninangiotensin-aldosterone system (RAAS) and renal vasoconstriction that contribute to increasing the extent of AKI among intensive care unit (ICU) patients [3, 4]. The incidence of AKI postoperatively comprises around 5-40% with a highly developed morbidity and mortality rates among patients and expands the hospitalization period [5, 6].
The etiologies of AKI could be one of the three potential scenarios; prerenal, renal (intrinsic) and postrenal. Prerenal AKI (prerenal azotemia) is characterized by renal hypoperfusion driven by systemic changes conferred by hypovolemia, hypotension, cardiac failure, liver cirrhosis, hepatorenal syndrome, sepsis, renal artery stenosis, renal vein thrombosis and antihypertensive drugs, leading to glomerular filtration rate (GFR) decline without affecting renal parenchyma. Renal (intrinsic) AKI depicts renal parenchymal damage due to glomerulonephritis, acute tubular necrosis, interstitial nephritis, and nephrotoxicity. Postrenal AKI is caused by urinary tract obstruction without parenchymal pathology [7].
However, the complexity surrounding AKI is that pathophysiological mechanisms could be explained as a result of hemodynamic changes including reduction of afferent arteriolar blood flow to the kidney and the mesangial contraction that aimed to minimize glomerular plasma flow, glomerular pressure, glomerular filtration surface area and finally leading to diminished GFR. This evidence could explain renal vascular resistance coming from disturbances in intrarenal vasoactive mediators through increasing vasoconstrictors (Angiotensin II, adenosine, thromboxane, leukotrienes, endothelin and platelet activating factor) against vasodilators (endothelial-derived nitric oxide and prostacyclin) contributing to persistent medullary hypoxia. As well, radiocontrast agents might reach this result by the same way [8, 9].
Furthermore, renal tubular injury and apoptosis contribute to reduced GFR by different ways: (1) Disruption of tubular reabsorption of NaCl and delivered to distal tubules would activate
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Doctoral Thesis
INTRODUCTION CHAPTER
tubuloglomerular feedback (TGF) mechanism through purinergic signaling in which macula densa sensor for NaCl “Na-K-2Cl cotransporter” (NKCC2) failed to discard higher concentrations of NaCl due to Na-K ATPase depletion leading to acid-base dysregulation and cell swelling. Hence, ATP isolated and hydrolyzed by 5`nucleotidase into adenosine molecule which is a potent vasoconstrictor to afferent arterioles and increases intracellular calcium leading to activation of proteases and endonucleases required for cell death according to Osswald’s hypothesis [10, 11].(2) Glomerular backleak of filtrates causes hypoproteinemia, interstitial edema due to albuminuria and increases the levels of blood urea nitrogen (BUN) and creatinine [12, 13]. (3) Renal tubular obstruction elevates renal tubular pressure and not the single nephron GFR (SN-GFR), but upset renal tubular recovery to potentiate cell apoptosis [14]. A general scheme of possible pathophysiological mechanisms of AKI was summarized in Figure (1).
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Doctoral Thesis
INTRODUCTION CHAPTER
Figure (1): A scheme of possible pathophysiological mechanisms of AKI. ǂ TGF: Tubuloglomerular feedback; ET: Endothelin; TX: Thromboxane; LT: Leukotrienes; PAF: Platelet activating factor; PGI2: Prostacyclin; EDNO: Endothelial-derived Nitric oxide.
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Doctoral Thesis
INTRODUCTION CHAPTER
The classification and definition of AKI are important to understand the chaos accompanied cardiac surgery and to establish a successful clinical diagnosis. Initially, the Acute Dialysis Quality Initiative (ADQI) consensus nominated RIFLE (Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease) as a classification criteria for AKI, dependent mainly on serum creatinine baseline which is difficult to be known clinically without Modification of Diet in Renal Disease (MDRD) equation, a specific for Chronic kidney Disease (CKD) only and/or Urinary Output (UO) [15]. Besides, serum creatinine is neither a specific nor sensitive marker and is affected by sex, age, dietary status , muscle mass, medications and raises lately after 50% of kidney function is lost [16, 17].
Another modifiable version of RIFLE criteria is the Acute Kidney Injury Network (AKIN) that reliably uses two values of serum creatinine and urinary output neglecting both GFR and baseline effects [18,19]. AKIN appended indispensable etiological information along with the ability for recognition and stratification of more AKI patients but failed to fulfill an improvement in the prognosis of hospitalized patient death due to slow and minimal changes in serum creatinine values over time [20]. Recently, an advanced tool for AKI classification, Kidney Disease Improving Global Outcomes (KDIGO) was validated for AKI staging and severity as it encompasses nearly all advantages of both RIFLE and AKIN criteria in a harmonized manner [21].
Previous studies proposed that increased predisposition of AKI after CPB in response to the effect of the machinery pump that lowers renal blood flow due to the absence of real pulsatility initiates an inflammatory sequence through stimulation of complement cascade with cytokine production and platelet aggregation leading to a probable ischemic renal injury [22, 23].
Cystatin C is 120 amino acids chain encoded from CST3 gene located in chromosome 20 p11.21 and considered to be an alternative of serum creatinine in GFR estimation, acting as an extracellular cysteine protease inhibitor present in almost every tissue, body fluids and massively rises up in response to inflammation and tissue injury without influence by non-renal factors [24, 25]. Kaseda et al mentioned that it was filtered by the glomerulus then reabsorbed in proximal tubules within megalin-mediated endocytosis and catabolized swiftly due to its short half-life that enables being reached easily to steady state [26].
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Doctoral Thesis
INTRODUCTION CHAPTER
After exposure to ischemic injury, GFR deteriorated rapidly and serum cystatin C increased within 48 h prior to any increase in serum creatinine. Despite, cystatin C is affected by fluid administration [27], steroid drugs, thyroid abnormalities and septicemia [28]. It was found that serum cystatin C is a better tool in the discrimination of AKI from pre-renal azotemia or nonAKI but not from CKD and a possible marker of AKI severity [29].
Cysteine-rich protein (CYR61) is a multifunctional heparin-binding protein encoded by CYR61 gene located on chromosome 1p22.3[30] and comprised of four different domains arranged from N to C termini as; Insulin-like growth factor binding protein (IGFBP) domain, von Willebrand type C repeats (vWC) domain, thrombospondin type 1 repeat domain (TSR), and C-terminal (CT) domain[31]. CYR61 may act as Wnt-1 proto-oncogene [32] and inducible-growth factor via binding with integrins promoting angiogenesis, chemotaxis, tumorigenesis and proliferation [33, 34]. Recent reports explained the stimulatory role of CYR61 towards proinflammatory cytokines like monocyte chemoattractant protein-1 (MCP1), Interleukin -6 (IL-6) and Tumor growth factor-β (TGF- β) that impose inflammation and tubulointerstitial fibrosis through recruitment of neutrophils and macrophages in early stages [35, 36]. Accordingly, Lai et al concluded that markedly decreased levels of urinary CYR61 after unilateral ischemic reperfusion injury (IRI) in mice might explain its ability to prevent consequences of inflammation accompanied with ischemic AKI [37]. Other studies showed that mRNA levels of CYR61 increased in proximal renal tubules and appeared in urine within 3−6 h after renal tubular injury preceding any increase in serum creatinine and peaked at 6-9 h, increasing the possibility to be a significant predictor for AKI in both preclinical and clinical studies [38, 39]. Unfortunately, urinary CYR61 reduced by time in spite of AKI progression and its quantitation proceeded by the poor insensitive immunoblotting method [40]. Thus, further investigations may introduce more information about diagnostic utility and possible therapeutic interventions.
Klotho was identified as an aging suppressor gene located on chromosome 13q12 [41] with various functions, encoded as a single transmembrane protein and expressed abundantly in distal tubules than proximal tubules [42]. The activation of klotho required membrane-bound proteases to remove the extracellular domain and immediately give rise in the blood, urine, and cerebrospinal fluid. Klotho may be present anchored to the membrane acting as a receptor for
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Doctoral Thesis
INTRODUCTION CHAPTER
fibroblast growth factor-23 (FGF-23) that regulate phosphate metabolism [43] and soluble klotho that is beneficial as an antioxidant [44], ion channels and growth factors activator [45, 46]. The molecular genetic analysis discovered six single nucleotide polymorphisms (SNPs) of klotho. Three of them present in introns region without any mRNA splicing, one at 1155 nucleotide number without any role in amino acid incorporation and the remaining two SNPs, F353V and C370S plays a pivotal role in survival and longevity over 75 years [47,48]. Despite, the mechanism of klotho role in AKI remains elusive. Previous studies revealed that renal and plasma levels of klotho declined drastically after 3h of ischemic renal injury till about 48 h and reverted to the baseline through a week. However, urinary levels of Klotho were tardily suppressed after 24 h and start to increase within 48 h of renal insult. Deeply, the reversible manner of klotho after marked reduction suggests that AKI is a provisional state of klotho deficiency [49] and proves the anti-apoptotic effect of klotho on renal tubular cells [50]. The diagnostic value of Klotho to predict AKI outcomes still needs to be explored in a large scale [51]. Indeed, the combination of possible and traditional biomarkers may markedly amplify the sensitivity for early detection of AKI. Prior to the study, we carried out a meta-analysis for previous papers with relevant interest in CYR61, klotho in particular and possible diagnostic utilities for AKI as an example for Evidence-based medicine (EBM) to constitute a comprehensive overview of the topic from all aspects, presents an empirical support from past conclusions and outcomes that could finally fortify making a correct decision. After a deep systematic search in the Medline, SCOPUS, ScienceDirect and Google Scholar by Mesh terms “Klotho and acute kidney injury” & “CYR61 and acute kidney injury”, only 11 papers were included in the meta-analysis; 4 for CYR61 and 7 for klotho. Thus, the effects found in the primary studies are converted into a standardized effect size which is no longer placed on the original measurement scale and can, therefore, be compared with measures from other scales.
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Doctoral Thesis
UVODNO POGLAVJE
UVODNO POGLAVJE Akutna ledvična okvara (ALO) je kritično epidemično prehodno stanje, ki se pojavi po operaciji srca kot ena od možnih etiologij. Za ALO je značilno hitro poslabšanje delovanja ledvic, ki pride do izraza kot povišanje ravni kreatinina v serumu v primerjavi z referenčno izhodiščno vrednostjo in/ali kot nenadno zmanjšanje izločanja urina. Število operacij srca se po vsem svetu vsako leto praktično podvoji, po navadi pa so take operacije povezane s kardiopulmonalnim obvodom (KPO), ki privede do zmanjšanega minutnega volumna, aktivacije renin-angiotenzin-aldosteronskega sistema (RAAS) in vazokonstrikcije v ledvicah. Vse to prispeva k povečanju obsega ALO pri bolnikih v enotah intenzivne nege (EIN). Pojavnost ALO po operaciji je približno 5–40 odstotkov, pri čemer se poveča obolevnost in umrljivost pri bolnikih ter podaljša obdobje hospitalizacije. Razvrstitev ALO z uporabo kriterijev KDIGO je bila potrjena za določanje stopenj in resnosti ALO, saj ta razvrstitev obsega skoraj vse prednosti kriterijev RIFLE in AKIN na usklajen način, s čimer se zmanjša vpliv kreatinina v serumu. Ta označevalec ni niti specifičen niti občutljiv, nanj vplivajo spol, starost, prehransko stanje, mišična masa in zdravila, zviša pa se šele, ko je izgubljenih že 50 % delovanja ledvic. Raven cistatina C v serumu se je kot odziv na zmanjšanje hitrosti glomerulne filtracije (HGF) dvignila v 48 urah pred kakršnimi koli spremembami ravni kreatinina v serumu. Zaradi nekaterih omejitev cistatina C se kaže potreba po določanju novih bioloških označevalcev, ki bi pokazali razvoj in prognozo ALO v zgodnjih fazah, kar bi lahko zvečalo učinkovitost zdravljenja in preprečilo neželene zaplete. Takšna označevalca bi lahko bila CYR61 in Klotho.
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Doctoral Thesis
AIM AND DESCRIPTION OT THE DOCTORAL THESIS CHAPTER
AIM AND DESCRIPTION OT THE DOCTORAL THESIS CHAPTER The overall aim of the doctoral thesis based on testing of these three main hypotheses: 1. Plasma/serum or urinary levels of Klotho and CYR61 are early prognostic biomarkers of AKI after cardiac surgery. 2. Klotho and CYR61 are markers of renal tubular dysfunction at the different levels of AKI after cardiac surgery. 3. The combinations of Klotho and CYR61 with other markers (f.e. NGAL/Klotho or KIM-1/Klotho ratios) will increase the prognostic efficiency of AKI after cardiac surgery.
The thesis is divided into seven independent chapters which are intercalated together and arranged rationally to form the final image based on elaborated topics. The “introduction chapter” gives a systematic review for acute kidney injury (AKI) after cardiac surgery, highlights biomarkers challenge toward early prediction of AKI and covers the following aspects:
Definition of AKI from general perspective and contribution of cardiac surgery with CPB.
Incidence of AKI after cardiac surgery.
Etiology and pathophysiology.
Classification of AKI by using newly KDIGO criteria with reminds by RIFLE and AKIN.
Biomarkers of renal function including serum creatinine and cystatin C with positive and negative contributions.
CYR61, klotho proteins and challenges to be competitive biomarkers with traditional ones.
Meta-analysis of results emerged from previous papers worked on CYR61 and klotho.
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Doctoral Thesis
AIM AND DESCRIPTION OT THE DOCTORAL THESIS CHAPTER
The “materials & methods chapter” provides a simple description for the practical work including subtitles: -
Studied population and criteria of selection.
-
Sample collection and storage.
-
Analytical methods and instrumentations.
-
Statistical tests used for analyzing data.
The “articles chapter” includes two scientific papers and one review article as follow: -
First scientific Paper: “Evaluation of serum cysteine-rich protein 61 and cystatin C levels for assessment of acute kidney injury after cardiac surgery” [ Published]
-
Second Scientific paper: “Serum Klotho as a marker for early diagnosis of acute kidney injury after cardiac surgery” [Submitted].
-
Review article: “Validity of Klotho, CYR61, and YKL-40 as ideal predictive biomarkers for acute kidney injury” [Accepted for publication].
The “results chapter” used to present data of the studied parameters involving; serum levels of creatinine, cystatin C, CYR61 and klotho after being statistically analyzed, well represented in texts, tables, figures and possible correlations between measured parameters were recognized without interpretation. As well, the diagnostic performance of these biomarkers was tested by ROC in which the concentration was plotted against time and AUCs were compared. Finally, meta-analysis data was represented by specific tables and figures.
All obtained results and their significance for the final outcome of the general research plan were assembled together, discussed, compared with results from previous and current analog studies in the “general discussion chapter”. The “conclusion chapter” includes final remarks of the thesis and prospective recommendations.
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Doctoral Thesis
NAMEN IN OPIS DOKTORSKE DISERTACIJE
NAMEN IN OPIS DOKTORSKE DISERTACIJE Glavni cilj doktorske disertacije temelji na testiranju teh treh glavnih hipotez: 1) Plazemska/serumska koncentracija Klotho in CYR61 je zgodnji napovedni dejavnik ALO po operaciji srca. 2) Klotho in CYR61 sta označevalca ledvične tubularne disfunkcije pri različnih stopnjah ALO po operaciji srca. 3) Kombinacija Klotho in CYR61 z drugimi označevalci (npr. razmerja NGAL / Klotho ali KIM-1 / Klotho) bo povečala napovedno vrednost za ALO po operaciji srca. Disertacija je razdeljena na sedem neodvisnih poglavij, ki so združena in smiselno razporejena, tako da tvorijo končno podobo na podlagi oblikovanih tem. »Uvodno poglavje« podaja sistematičen pregled za akutno ledvično okvaro (ALO) po operaciji srca, izpostavi izziv bioloških označevalcev pri zgodnjem napovedovanju ALO in pokriva naslednje vidike:
Opredelitev ALO s splošnega vidika in prispevek operacije srca s KPO.
Pojavnost ALO po operaciji srca.
Etiologija in patofiziologija.
Razvrstitev ALO z uporabo novih kriterijev KDIGO, ki združujejo kriterije RIFLE in AKIN.
Biološki označevalci ledvične funkcije, vključno s kreatininom v serumu in cistatinom C s pozitivnimi in negativnimi lastnostmi.
Proteina CYR61 in klotho ter izziv pri njunem uveljavljanju kot novejša biološka označevalca v primerjavi s tradicionalnimi.
Meta-analiza rezultatov iz prejšnjih člankov, ki so se ukvarjali s proteinoma CYR61 in klotho.
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NAMEN IN OPIS DOKTORSKE DISERTACIJE
Poglavje »Materiali in metode« podaja preprost opis praktičnega dela, vključno z naslednjimi podnaslovi: -
Opazovana populacija in merila za izbor.
-
Zbiranje vzorcev in shranjevanje.
-
Analitske metode in instrumenti.
-
Statistični testi, uporabljeni za analizo podatkov.
Poglavje »Članki« vključuje dva znanstvena prispevka in en pregledni članek: -
Prvi znanstveni prispevek: "Vrednotenje ravni beljakovine CYR61 in cistatina C v serumu za ocenjevanje akutne ledvične okvare po operaciji srca" [Objavljeno].
-
Drugi znanstveni prispevek: " Klotho v serumu kot označevalec za zgodnjo diagnozo akutne ledvične okvare po operaciji srca " [Predloženo].
-
Pregledni članek: "Veljavnost Klotho, CYR61 in YKL-40 kot idealnih napovednih biooznačevalcev za akutne ledvične okvare " [Sprejeto za objavo].
Poglavje »Rezultati« prikazuje podatke o obravnavanih parametrih – raven kreatinina in cistatina C ter proteinov CYR61 in Klotho v serumu – po statistični analizi. Podatki so predstavljeni v besedilu, preglednicah, slikah, možne korelacije med merjenimi parametri pa je bilo mogoče prepoznati brez razlage. Diagnostično delovanje teh bioloških označevalcev je bilo preizkušeno s krivuljo ROC, pri čemer je bila koncentracija podana skupaj s časom, primerjali pa smo mere AUC. Na koncu so podatki meta-analize predstavljeni v posebnih preglednicah in slikah. Vse dobljene rezultate in njihov pomen za končen izid splošnega raziskovalnega načrta smo združili ter jih obravnavali in primerjali z rezultati iz prejšnjih in trenutnih analognih študij v poglavju »Splošna razprava«. Poglavje »Sklepne ugotovitve« vključuje končne pripombe disertacije in potencialna priporočila.
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MATERIALS & METHODS CHAPTER
MATERIALS & METHODS CHAPTER 1.1 Study design The study was conducted according to the declaration of Helsinki. After approval of the study protocol by “The National Medical Ethics Committee of the Republic of Slovenia”, informed consents were obtained from all study participants prior to data collection. Our study was carried out on fifty patients of matched age and sex underwent for cardiopulmonary bypass (CPB) in The University Medical Center Ljubljana for cardiac surgery. They were divided in accordance with KDIGO criteria in Table (1)[21] into AKI group and non-AKI group. The inclusion criteria encompassed ages from 20 – 80 years old and patients who suffered from progressive cardiac problems. Otherwise, patients with a history of kidney diseases, chronic autoimmune diseases, malignancies, diabetic nephropathy, overlapping syndromes, renal transplants, and pregnants were excluded. Additionally, a proactive meta-analysis for previous papers focused on CYR61 and klotho was done to present full understanding for tolerable effects on studies outcomes.
Table (1):KDIGO criteria for classification of acute kidney injury (AKI) patients[21]. Stages 1
Serum creatinine criteria Serum creatinine increase ≥26.5 μmol/l within 48 hours.
Urine output criteria 2.0–2.9-fold from baseline.
3.0-fold from baseline.
2-3 fold from baseline or urinary output < 0.5 ml/kg/h for ≥ 12 h.
Increase in serum creatinine >2.0 to 2.9 fold from baseline or urinary output < 0.5 ml/kg/h for ≥ 12 h.
Increase in serum creatinine ≥ 3 fold from baseline or serum creatinine ≥ 354 μmol/l or initiation of renal replacement therapy or urinary output < 0.3 ml/kg/h for ≥ 24 h or anuria ≥ 12 h.
Increase in serum creatinine ≥ 3 fold from baseline or serum creatinine ≥ 353.6 μmol/l (˃ 4 mg/dl) or start of RRT or urinary output < 0.3 ml/kg/h for ≥ 24 h or anuria ≥ 12 h.
Stage III
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ARTICLES CHAPTER CHAPTER 3/ POGLAVJE 3
Table 2. Outlines of the search strategies used for each database. Number of papers yielded per searchable database
PubMed
Klotho AND "acute kidney injury"[MeSH Terms]
31
Cysteine rich protein 61 and “acute kidney injury”
11
Chitinase-3-like protein 1 and “acute kidney injury”
4
Klotho and “ acute kidney injury”
45
CYR61 and “ acute kidney injury”
9
Chitinase-3-like protein 1 and “acute kidney injury”
5
Klotho and AKI/“acute kidney injury”
2
CYR61
1
YKL-40
2
Klotho
21
CYR61
4
Chitinase-3-like protein 1
6
Klotho
8
CYR61
1
YKL-40
2
Klotho biomarker and “acute kidney injury”
256
CYR61 and “acute kidney injury”
108
YKL-40 and “acute kidney injury”
77
Klotho and AKI
909
SCOPUS
SciELO
Cochrane Library
Lilacs
Science Direct
Google Scholar
CYR61 biomarker and "acute kidney
Number of Inclusions
663
Number of Exclusions
Excluded because of duplication or lack of match with specialization of proposed description
Search strategy
Included in the review article
Database used
injury" YKL-40 biomarker and acute kidney injury
752
2917
Total
72
17
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ARTICLES CHAPTER CHAPTER 3/ POGLAVJE 3
Table 3. Summary of characteristics and main results of the 17 previous studies included in this review. Serials
Author /year
Study design
Purpose of the study
Results and recommendations
1
De Loor et al.,37
Pilot study
To evaluate whether urinary Chitinase 3-like protein 1 (YKL40) can predict AKI stage ≥ 2 in ICU patients compared with NGAL.
The concentration of UCHI3L1 within 12 hours of AKI stage ≥ 2 was increased with good performance on AUC-ROC curve (0.792, 95% CI), similar to UNGAL AUC-ROC (0.748, 95% CI), and after 24 h, UCHI3L1 showed AUCROC twice as high (95% CI: 1.3–3.1) as controls.
2
Huen et al.,38
Review
3
Schmidt et al.,39
Cohort (comparative) study
Focus on future phenotyping of AKI regarding NGAL and YKL40. To evaluate the role of urinary and blood levels of YKL-40 in allografts after renal transplantation.
NGAL and YKL-40 are important novel biomarkers involved in moderate renal tubular protection after AKI. Urinary YKL-40 increased early on, within 18 h after surgery (131.3 ± 155.2), with AUC 0.86 ± 0.07; blood YKL-40 retarded to 1 day after surgery (623 ± 285.9), with AUC 0.59 ± 0.08
4
Hall et al.,40
Observational cohort study
To measure YKL-40 levels in the urine of clinically hospitalized AKI patients.
Urinary YKL-40 levels were detectable (≥ 5 ng/ml) within 1 h and gave better prognostic value (P = 0.04) with NGAL.
5
Tatar et al.,41
Cohort study
6
Maddens et al.,36
Clinical and experimental study
To define relationship between Mean serum YKL-40 and proteinuria YKL-40 and proteinuria in renal levels were 66 ± 46 ng/ml and 0.77 ± transplant recipients. 1.15 g/day respectively without any apparent correlation. Measurement of urinary and Urinary CHI3L1 higher in septic-AKI plasma levels of Chitinase 3-like patients than in non-AKI (P ˂ 0.05). But protein 1 and -3 in mice and in septic-AKI mice models, CHI3L1 and patients with and without septic -3 were found to be high. AKI.
7
Malyszko et al.,42
Review article
Illustration of candidate Elevated YKL-40 in both urine and biomarkers in cases of delayed serum levels of patients with DGF, 2 graft function as a form of acute days after transplantation. kidney injury.
8
Muramatsu et al.,32
Experimental study
To test CYR61 in the urinary CYR61 protein increased first within 1 h levels of mice and rats after and appeared in urine 3-6 h after immediate renal ischemic ischemic renal injury. reperfusion injury.
9
Lai et al.,43
Experimental study
To investigate the role of CYR61 CYR61 was significantly induced at after unilateral IRI in mice. renal and urinary levels after IRI.
10
Xu et al.,44
Experimental study
To indicate CYR61 expression in Enhanced expression of renal CYR61 in renal cell lines under hypoxia response to hypoxic ischemic injury.
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11
Kim et al.,45
Cohort study
To determine possible influence of AKI on serum and urinary levels of Klotho, S100A8/A9 and NGAL
Urinary Klotho levels were 13.21 ± 17.32 versus 72.97 ± 17.96 pg/ml (P = 0.002) in pre-renal and intrinsic AKI respectively.
12
Torregrosa et al.,46
Cohort study
Assessment of urinary Klotho Klotho levels did not behave as a good levels in patients after cardiac early biomarker of AKI. surgery or coronary angiography.
13
Castellano et al.,47
Experimental study
To investigate whether or not Complement activation result in complement components affect remarkable decline in renal Klotho Klotho levels after IRI. levels, 24 h after IRI.
14
Liu et al.,48
Case-control study
To evaluate serum Klotho levels Serum Klotho levels were 101.97 ± at different time intervals after 16.93 versus 121.64 ± 19.87 (P < 0.01) cardiac surgery. in AKI and non-AKI group respectively at 0 h and continued until 4 h. After 3 days, serum Klotho values were 120.50 ± 13.17 versus 128.67 ± 18.84.
15
Seo et al.,49
Retrospective cohort study
Assessment of renal Klotho Renal Klotho levels were significant levels in human samples instead reduced with AKI severity. of animal models.
16
Hu et al.,29
Experimental and casecontrol study
To estimate Klotho at urinary and Urinary Klotho values (pmoles/l) were plasma levels, investigating 2.52 ± 0.76 in AKI versus 20.66 ± 1.81 probable protective ability. in non-AKI, with P < 0.01.
17
Sugiura et al.,30
Experimental study
To explain the physiological Renal Klotho levels were significantly relevance of renal Klotho after reduced in IRI rats, 24 h after ischemia. IRI in rats.
Table 4. Description of biomarkers, their functions and measurement methods. Biomarker
Description
Encoded gene
Renal function
Detection sites
Measurement method
KLOTHO
Type I transmembrane protein
KL gene
Renoprotective and antiapoptotic
Kidney
PCR
Blood
ELISA
Urine
Immunoblotting
Kidney
PCR
Blood
ELISA
Urine
Immunoblotting
Kidney
PCR
Blood Urine
ELISA Immunoblotting
CYR61
YKL-40
Matricellular protein (Angiogenic factor)
CYR61 gene
Secreted glycoprotein (anti-apoptotic)
CHI3L1 gene
Cell proliferative and anti-apoptotic Inflammatory
* PCR = polymerase chain reaction; ELISA = Enzyme –Linked Immunosorbent Assay.
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RESULTS CHAPTER
RESULTS CHAPTER Fifty patients who met the inclusion criteria and classified according to KDIGO criteria, 26 of the patients developed AKI within 48 h after cardiac surgery and were nominated in the AKI group and the remaining 24 patients without AKI and were placed in the non-AKI group as shown in Table (2). Table (2): A synopsis of clinical and demographic data from all study participants.
Parameters Number of patients and Gender (m/f) Age (yrs.) BMI (kg/m2) Euroscore II Diabetes mellitus (non/oral/insulin) Arterial hypertension (y/n) Left Ventricular Ejection Fraction (%) Preoperative Creatinine (µmol/L) Preoperative eGFR (ml/min/1.73 m2) eGFR ≤ 60 ml/min/1.73 m2 (number of patients) Intraoperativedata CPB time (min) RBC units / transfusion ( ml/unit) Fresh frozen plasma /transfusion ( ml/kg) Platelets transfusion ( ml/unit)
Non-AKI
AKI
Test values
P-value
24 (12/12) 79.86 ± 10.9 27.26 ± 4.4 2.2 ± 3.1 20/2/2 18/6 59.76 ± 16.1 64.8 ± 17.9 95.16 ± 22.1 1
26 (18/8) 70.16 ± 13.4 27.36 ± 4,7 2.7 ± 1.6 15/7/4 24/2 59.26 ± 7.3 74 ± 43.3 93.76 ± 34.1 6
1.92a 0.094b 0.08b FETc 4.06a 2.78a 0.18b 0.39b 0.78b FETc
0.409 0.770 0.841 0.480 0.448 0.520 0.718 0.947 0.531 -----
96.8±33.6 2.2 ± 3.0 1.9 ± 3.0 0.19± 0.40
101.8 ± 37.1 2.5 ± 3.3 1.5 ± 2.6 0.25 ± 0.53
3.22b 2.11b 0.84b 0.466b
0.001 0.01 0.495 0.641
14.7 ± 16.1 30.2 ± 58.1 10.3 ± 11.6 18.6 ± 24.9
14.5 ± 16.8 33.4 ± 54.1 4.8 ± 3.8 12.2 ± 7.2
0.268b 0.412b 1.01b 0.366b
0.767 0.618 0.469 0.659
Postoperative data Respiratory support (h) Inotropes (h) ICU stay (days) Hospital stay (days)
ǂ Data are represented as mean ± SD; BMI: Body Mass Index; eGFR: Estimated Glomerular Filtration Rate; CPB: Cardiopulmonary bypass; ICU: Intensive Care Unit. ǂǂa. Chi-square test; b. Student-t-test; c. FET: Fisher's exact test.
Regarding the preoperative levels of serum creatinine was shown to be elevated in the AKI group (74.06 ± 43.29 µmol/L) than in the non-AKI group (64.86± 17.90 µmol/L). Immediately after cardiac surgery, serum creatinine values were significantly decreased in the non-AKI group (62.06 ± 16.43 µmol/L) compared with the AKI group (83.36 ± 28.1 µmol/L). After 2 hours, a significant raise noticed (93.36 ± 38.69µmol/L) and continued to reach a plateau after 48 hours (124.66 ± 67.22 µmol/L) in the AKI group. However, the non-AKI group exhibited a slight increase in serum creatinine within 2 hours (69.76 ± 14.3) and abruptly fall down upon 2 days to be near the baseline levels (64.66 ± 22.4). Moreover, changes in serum creatinine levels over time in both studied groups were represented in Figure (2). Likewise, serum creatinine was found to be directly significant correlated with CYR61 and cystatin C in the preoperative period and with only cystatin C in both time intervals (0h, 2h) as in Figure (3).
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Figure (2): A plot of serum creatinine levels (µmol/L) in AKI and non-AKI groups at different time intervals of CPB.
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(A)
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(B) Figure (3): (A) Correlations of serum creatinine with CYR 61andcystatin C values before the CPB. (B) Correlations of serum creatinine with cystatin C values at (0h and 2h) of CPB.
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Serum cystatin C levels in the AKI group started to increase significantly after 2 hours and multiplied within 48 hours (1421.6 ± 739µg/L) with p < 0.001 compared with the initial baseline within 2 hours (949.6 ± 557 µg/L) with p < 0.001. While in the non-AKI group it was noticed to be (700.6 ±170 µg/L) with p < 0.001 within 2 hours and persisted in increasing up to (910.6 ± 422 µg/L) with p< 0.001 after 48 hours. Serum cystatin C concentrations were plotted against time as in Figure (4).
Figure (4): A plot of serum cystatin C concentrations (µg/L) in AKI and non-AKI groups at different time intervals of CPB.
Upon checking of CYR61 data by using normality test, we showed that the distribution was distorted from the normal curve and recommended to be expressed as median values with interquartile ranges. The initial median values of serum CYR61 concentrations were overexpressed in the non-AKI group (20.35 µg/L) than in the AKI group (12.35 µg/L). By the time, serum CYR61 values dropped simultaneously in both groups within 2 days with a bit increase in the AKI group than the non-AKI group and represented by Figure (5). Surprisingly, serum CYR61 and cystatin C associated together at 2 time intervals (before, after 2h) and with serum creatinine at 0h, after 2 h and 1 day postoperatively as in Figure (6).
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Figure (5): Serum CYR 61 values (median and interquartile ranges) at five time-points: before surgery, at the end of CPB and 2h, 24h and 48h after the end of CPB.
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(A)
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(B)
Figure (6): A.Correlations between serum CYR 61 and cystatin C before and after 2 h of CPB; B. Correlations between serum CYR 61 and creatinine levels at 0 h, after 2h and 24 h of CPB.
Before cardiac surgery, serum klotho levels (µg/L) were found to be superior in the AKI group (1.69) than in the non-AKI group (1.40). Then, its levels were subjected to abrupt reduction in both groups immediately after cardiac surgery. Nevertheless, klotho levels (µg/L) within 2 hours retrieved increasingly than the baseline in AKI group (2.32), non-AKI group (1.79) and ultimately decreased within 48 hours as in Table (3). For understanding the behavior of klotho levels in AKI and non-AKI groups, the changes in serum klotho values were plotted against time in Figure (7). As well, levels of serum klotho and klotho/creatinine ratio were positively correlated in 2h and 24 h postoperatively as in Figure (8). The performance of all measured parameters was assessed after 2 h of CPB by using ROC curve and the resultant AUCs of serum creatinine, cystatin C, CYR61, klotho and klotho/creatinine ratio were 0.767, 0.655, 0.513, 0.653 and 0.547 respectively as shown in Figure (9).
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The logistic multivariable regression analysis showed thatpreoperative cystatin C levels were independently associated with postoperative levels of (cystatin C, CYR61 and klotho) and might be a potential risk factor for AKI as in Table (4).
Table (3): Serum Klotho levels (µg/L) in AKI and non-AKI groups at different points after CPB. Non-AKI group
AKI group
t- test values
P value
N=24
N=26
Pre Klotho (µg/L)
1.405
1.69
0.401
0.636
Klotho 0h (µg/L)
0.470
0.545
0.399
0.740
Klotho 2h (µg/L)
1.795
2.325
0.674
0.261
Klotho 24h (µg/L)
1.21
1.77
0.822
0.220
Klotho 48h (µg/L)
1.21
1.255
0.711
0.534
K
5.06
4.85
P
0.0368*
0.042*
ǂ Median values are represented in the table.Pre: before surgery; 0h: at the end of CPB; 2h, 24 and 48h: hours after the end of CPB. ؞Kruskal Wallis test with post hoc for testing the significance between different time intervals within each group, using Duncan’s method by using small letters, [the same small letters indicate that there was no significant difference, while the larger letters indicate that there was a significant difference]. * Significance at p≤0.05.
Figure (7):Comparison of serum Klotho values (µg/L) in AKI and non-AKI groups at different time intervals.
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Figure (8): Correlations between serum klotho and klotho/creatinine ratio after 2 h and 1 day of CPB.
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Figure (9): ROC curve for serum creatinine, CysC, CYR61 and klotho values 2h after the end of CPB.
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Table (4): Multivariate logistic regression model of different studied parameters and its effect on dependent variables.
Dependent variable 0 h Cystatin C(µg/L)
2 h Cystatin C(µg/L)
0 h CYR61(µg/L)
2 h CYR61 (µg/L)
24 h CYR61 (µg/L)
0 h Klotho(µg/L)
Independent variable
OR
95% CI
t-test
p
Pre Creatinine (µmol/L)
0.7
0.65-1.11
0.916
0.210
Pre Cystatin C (µg/L)
2.31
0.25-0.69
2.36
0.012*
CBP time ( min)
0.78
0.61-1.15
1.045
0.110
Pre Creatinine (µmol/L)
0.64
0.21-1.07
1.22
0.152
Pre Cystatin C (µg/L)
1.98
0.32-0.87
2.19
0.0229*
CBP time ( min)
0.59
0.31-1.259
1.043
0.230
Pre Creatinine (µmol/L)
0.4
0.31-1.259
1.09
0.240
Pre Cystatin C (µg/L)
2.36
0.21-0.71
3.69
0.001*
CBP time ( min)
0.87
0.42-1.202
1.089
0.230
Pre Creatinine (µmol/L)
0.79
0.41-1.02
1.364
0.130
Pre Cystatin C (µg/L)
2.56
0.12-0.72
3.14
0.001*
CBP time ( min)
0.85
0.39-0.979
0.946
0.190
Pre Creatinine (µmol/L)
0.44
0.74-1.368
0.946
0.120
Pre Cystatin C (µg/L)
1.94
0.21-0.49
2.85
0.021*
CBP time ( min)
0.63
0.79-1.038
1.344
0.200
Pre Creatinine (µmol/L)
0.37
0.9-0.958
1.665
0.090
Pre Cystatin C (µg/L)
1.87
0.36-0.811
1.98
0.033*
CBP time ( min)
0.72
0.4-1.352
1.023
0.380
*significance at p< 0.05. Pre: before surgery; 0 h: at the end of CPB; 2 and 24hours after the end of CPB.
For Met-analysis study, the effect sizes for all included studies calculated byCohen’s d summarized in Table (5) and represented by both random effect model and funnel plot as shown in Figures (10, 11) respectively.
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Table (5): Results from the random effects of Meta-analysis.
1 2 3 4 5 6 7 8 9 10 11 Total (fixed effects) Total (random effects)
Estimate (Log)
SE
Estimate
95% CI
Z
P
5.329 1.737 -3.635 0.481 0.000 4.170 -2.030 1.637 8.404 5.846 8.059 0.934
0.333 0.150 0.264 0.165 0.251 0.431 0.306 0.342 1.396 0.484 1.143 0.081 6 0.806
65 100 72 70 32 33 30 20 10 40 10 482
4.670 to 5.987 1.441 to 2.033 -4.156 to -3.114 0.154 to 0.807 -0.502 to 0.502 3.310 to 5.030 -2.641 to -1.419 0.949 to 2.326 5.471 to 11.338 4.884 to 6.807 5.710 to 10.409 0.774 to 1.094
11.451
