Validity of Klotho, CYR61 and YKL-40 as ideal predictive biomarkers for acute kidney injury: review study

ABSTRACT CONTEXT AND OBJECTIVE: Acute kidney injury (AKI) is still a headache for clinicians and scientists as a possible reason for increased death among intensive care unit (ICU) patients after invasive cardiac surgery. Furthermore, the diagnostic process for AKI using conventional biomarkers is not sufficient to ensure early warning of this condition because of the morbid influence of non-renal factors that definitively delay the time for the prognosis. These imposed limitations have led to significant amounts of research targeted towards identifying novel biomarkers for AKI with a sustained degree of sensitivity and specificity. Here, we reviewed previous studies conducted on the Klotho, CYR61 and YKL-40 biomarkers in relation to AKI. DESIGN AND SETTING: Review of the literature conducted in the Institute of Clinical Chemistry & Biochemistry, Ljubljana University Medical Center, Slovenia. METHODS: The literature was searched in PubMed and the Cochrane Library. From the database of this specialty, we selected 17 references that matched our context for detailed analysis and further investigation. RESULTS: The studies reviewed showed notable differences in their results relating to the diagnostic impact of Klotho, CYR61 and YKL-40 on early prediction of AKI. CONCLUSIONS: The results regarding the Klotho, CYR61 and YKL-40 biomarkers showed markedly equivocal performance in the previous studies and did not fulfill the expectations that these factors would form valid possible biomarkers for AKI.


INTRODUCTION
Acute kidney injury (AKI) is a highly progressive critical problem that often occurs after invasive cardiac surgery using cardiopulmonary bypass (CBP). 1,2It threatens the life of intensive care unit (ICU) hospitalized patients through accompanying irreversible adverse outcomes that ultimately contribute to a 60% increase in mortality rate. 3Defining AKI is dependent on measurement of baseline serum creatinine, the traditional biomarker of kidney function, which remains unchanged until a sudden 50% of kidney function is lost. 4Moreover, AKI has been found to be strongly affected by dietary status, exercise, protein supplements, corticosteroids, age, gender and muscle mass. 5,6Therefore, there is an urgent need for novel biomarkers to predict and diagnose AKI at its earlier stages, so as to prevent complications and potentiate therapeutic approaches.

Classification of AKI
The Acute Dialysis Quality Initiative Group (ADQI) meeting in 2004 gave rise to a new regular criterion for analyzing kidney function, termed Risk Injury Failure Loss of function and End stage (RIFLE). 7,8RIFLE was dependent on serum creatinine (SCr) or urinary output (UO) measurements to determine the prognostic severity of deterioration of kidney function, classified into three stages. 8Many studies mentioned that the usefulness of RIFLE was affected by the following substantial limitations: [1] calculation of the SCr baseline using the Modification of Diet in Renal Disease (MDRD) equation showed high specificity for chronic kidney disease (CKD) but not AKI; [2] SCr was directly influenced by nonspecific factors and hence was unreliable; [3] using UO was a good alternative for SCr, but it was affected by diuretics and could only be measured by using a bladder catheter in an ICU and not among long-stay hospitalized patients; and [4] SCr was considered to be a marker for renal function, not kidney injury. 9bsequently, a modified standard was published in 2007 under the name "Acute Kidney Injury Network (AKIN)", with the aim of closing gaps generated by RIFLE.AKIN used two values of SCr within two days instead of baseline SCr, regardless of glomerular filtration rate (GFR) changes.According to AKIN, stage 3 AKI was confirmed when the duration of increased SCr levels did not exceed 48 h and the patient required renal replacement therapy (RRT). 10e failure of both RIFLE and AKIN to fulfill precise prognostic stratification of AKI severity and to provide a unified definition of AKI was the reason for establishing the Kidney Disease Improving Global Outcomes (KDIGO) guidelines.These novel criteria suggested that AKI should be defined by SCr levels that reached 26.4 μmol/l within 48 h or increased to a level 1.5 times higher than the baseline level within 7 days, which provides a sufficient timeframe for AKI diagnosis. 11The differences between all the diagnostic criteria are summarized in Table 1.

Epidemiology of AKI
3][14] Recent studies conducted in the USA and Spain showed incidences of approximately 23.8 cases per 1000 discharges and 209 cases per million, respectively. 15,16A recent population-based study conducted in the UK reported high incidence of AKI, of 1811 cases per million in 2003. 17A report from Kuwait indicated an incidence of 4.1 cases per 100,000 population per year. 18In addition, the annual incidences for AKI in Brazil and northern India were 7.9 and 6.4 cases per 1000 admissions. 19,20Notably, the mortality

Stage I
Increase in serum creatinine ≥ 1.5 times from baseline or decrease in estimated glomerular filtration rate ≥ 25% or urinary output < 0.5 ml/kg/h for ≥ 6 h.
Increase in serum creatinine ≥ 26.4 μmol/l or increased 1.5 to 2 fold from baseline or urinary output < 0.5 ml/kg/h for ≥ 6 h.
Increase in serum creatinine ≥ 26.5 μmol/l or increased 1.5 to 1.9 fold from baseline or urinary output < 0.

Klotho
Klotho (KL) is a novel phosphatonin encoded by the anti-aging KL gene located on chromosome 13q12 as an inactive singlepass transmembrane protein. 22Upon activation through action by membrane bound-secretases like ADAM10 and ADAM17, driven by insulin, the extracellular domain is cleaved and its serum, urine and cerebrospinal fluid levels become elevated. 23is ectodomain was termed a soluble Klotho, which would possibly bind directly with FGFR and tend to form an active complex exhibiting high affinity against FGF, 24 thereby alleviating oxidative stress through suppression of growth factors and stimulation of calcium ion channels (TRPV5 and TRPV6) 23 and potassium channels (ROMK) 25 but not sodium-phosphate cotransporters. 26Meanwhile, the remaining membrane Klotho would function as a coreceptor for bone regulatory hormone FGF23. 27Normally, Klotho shows greater expression in distal rather than proximal convoluted tubules in the kidneys, and in the choroid plexus of the brain rather than in the heart and parathyroid gland. 28e pathological importance of Klotho emerged through studies on animal models for AKI that had previously undergone ischemic reperfusion injury (IRI) or unilateral urethral obstruction (UUO).Thus, a transient reduction in renal Klotho mRNA expression was shown in response to renal tubular injury. 29,30 ther studies on Klotho applied to humans have demonstrated that the urinary and plasma levels of Klotho in patients with AKI are notably lower than in healthy individuals. 29From these observations, it has been proposed that Klotho has a role in exacerbating renal damage and has potential as a likely biomarker for AKI.

Cysteine-rich protein 61 (CYR61)
CYR61 is a cysteine-rich matricellular protein encoded by the CYR61 gene located on chromosome 1p22.3.It is intercalated with various integrins and heparin sulfate proteoglycans and is associated with extracellular matrix formation, cell adhesion, proliferation, differentiation, angiogenesis, apoptosis and inflammation due to its biochemical features, which resemble Wnt-1 proto-oncogene, and its number of growth factors. 31ditionally, renal CYR61 mRNA and protein expression, along with urinary levels, have been found to increase in IRI animal models that suffered from significant hypoxia, despite being indistinguishable at renal levels in normal tissues. 32,33is result provides encouragement to study CYR61 levels in humans, in order to elucidate its preventive and/or predictive role against AKI.

Chitinase-3-like protein 1 (YKL-40)
Chitinase-3-like protein 1 (CHI3L1) or YKL-40 is a 40 kDa glycoprotein 34 that is expressed from the CHI3L1 gene located on chromosome 1q31-q32. 35It is considered to be a member of the family of 18 glycoside hydrolases that encompasses chitinases but without any enzymatic activity.It is secreted by various cell types, including macrophages, chondrocytes and some types of cancer cells. 34Furthermore, Johansen et al. revealed that YKL-40 increased inflammation through activation of the innate immune response and regulation of tissue remolding. 35In addition, Maddens et al. collected urine samples from mice that presented sepsis two days after intrauterine injection of E. coli, and from human patients with sepsis.They showed similar quantitative increases in comparison with controls without AKI. 36erefore, studies on YKL-40 remain a prerequisite for understanding the pathophysiology of AKI.

OBJECTIVE
The objective of the current review was to focus on the suitability and validity of Klotho, CYR61 and YKL-40 as ideal predictive biomarkers for acute kidney injury.

METHODS
We conducted a comprehensive systematic search by using the main known databases: PubMed, SCOPUS, SciELO, Lilacs, ScienceDirect and Google Scholar.The MeSH search terms included: (''Klotho and Acute Kidney Injury''), (''CYR61 and Acute Kidney Injury'') AND (Chitinase-3-like protein 1 and Acute Kidney Injury'').The search strategy was designed for the PubMed database and was altered as needed for use in other databases.Our last search was finished in January 2016.
References were written in the English language.The inclusion criterion was that all research articles, review articles and observational studies included needed to match our context, i.e. "the propensity of CYR61, Klotho and YKL-40 to be novel biomarkers for AKI".Additionally, we excluded papers that investigated these biomarkers in relation to chronic kidney disease (CKD) and other diseases as well as AKI.

DISCUSSION
In this review article, we discuss the propensity of some novel biomarkers for early detection of AKI.Traditional biomarkers have been proven to be unable to satisfactorily distinguish AKI during the first 24 hours before kidney function is disrupted.This is certain to delay the diagnostic process and gives rise to the possibility that the patient's condition will worsen.Despite the paucity of studies on biomarkers and AKI (for reasons mentioned earlier), we conducted a comprehensive review of the literature encompassing all papers relating to our context, focusing on all the results.
1][52] Furthermore, Hu et al. observed that Klotho levels in both plasma and urine declined immediately in AKI animal models and were detectable within 3 h after injury.This change preceded any changes in serum creatinine by 1 day and plasma NGAL by 5 h, thus suggesting that Klotho may be an early biomarker for renal parenchymal injury. 29,53In the same manner, Kim et al. demonstrated that there were lower urinary Klotho levels in patients with pre-renal AKI than those with intrinsic AKI, and that this was not accompanied by any change in NGAL at the serum and urinary levels. 45giura et al. indicated that renal Klotho levels in rats started to fall on the first day and completely returned to normal within 10 days. 30

Experimental study
To test CYR61 in the urinary levels of mice and rats after immediate renal ischemic reperfusion injury.
CYR61 protein increased first within 1 h and appeared in urine 3-6 h after ischemic renal injury.9 Lai et al. 43 Experimental study To investigate the role of CYR61 after unilateral IRI in mice.
CYR61 was significantly induced at renal and urinary levels after IRI.Renal Klotho levels were significant reduced with AKI severity.16  Hu et al. 29

Experimental and casecontrol study
To estimate Klotho at urinary and plasma levels, investigating probable protective ability.
Urinary Klotho values (pmoles/l) were 2.52 ± 0.76 in AKI versus 20.66 ± 1.81 in non-AKI, with P < 0.01.17 Sugiura et al. 30 Experimental study To explain the physiological relevance of renal Klotho after IRI in rats.
Renal Klotho levels were significantly reduced in IRI rats, 24 h after ischemia.
and non-AKI patients after cardiac surgery or coronary angiography, thus dismissing the possibility that Klotho would be a sensitive AKI biomarker. 46 gery.An emerging suggestion to use the SCr/KL ratio instead of serum creatinine or Klotho alone could improve their diagnostic sensitivity for AKI at later times. 48udies on Klotho were found to exhibit a variety of problematic issues: almost all the studies related to animal models rather than humans, with a narrow scale; there were unexplained variations between comparable studies; the mechanism of Klotho in AKI remains unknown, the behavior of Klotho in animal models differed from its behavior in humans; there was a lack of knowledge of ideal Klotho timing and normal cutoff ranges; and the urinary and plasma levels of Klotho were not indicative for renal Klotho, which might suggest that confounding factors and discrepancies in laboratory methodologies were present.
According to Vaidya and Muramatsu et al., CYR-61 was rapidly stimulated in the proximal renal tubules and was excreted in urine within 3-6 h after bilateral renal ischemic injury in rats.Its peak was within 6-9 h and it declined after 24 h. 32,54Consequently, urinary CYR61 might act as an acceptable biomarker and screening tool for AKI, with follow-up in both preclinical and clinical studies. 32,55Moreover, Lai et al. conducted experiments on mice that proved that proinflammatory TGF-β enhanced renal CYR61 in mRNA and protein levels within 10 days after occurrences of unilateral ureteral obstruction (UUO). 56Subsequently, CYR61 gave rise to inflammatory sequelae through activation of monocyte chemoattractant protein-1 (MCP-1), thereby leading to monocyte chemotaxis and macrophage infiltration. 57This evidence revealed that inhibition of CYR61 could prevent adverse consequences that would contribute towards irreversible AKI-CKD transition, through postponing inflammation, tubulointerstitial fibrosis and apoptosis. 43 Akt and ERK signaling pathways. 44her previous papers investigating CYR61 expression found that it was induced by several growth factors, exposure to UV irradiation, 58 hypoxic conditions, vigorous exercise, 59 bacterial infections 60 and viral infections. 61Likewise, Pendurthi et al. mentioned that clotting factor VIIa (FVIIa) and thrombin triggered CYR61 redundancy, forced through blood coagulation. 62This observation matched with Hviid et al., who indicated that CYR61 levels increased at sites of surgical wound closure and that CYR61 was absent from systemic blood, which might explain the mediatory role of platelets in accumulations of CYR61 at sites of tissue injury in AKI patients. 63he diagnostic capacity of urinary CYR61 as a biomarker might be blocked through: 1) its poor specificity, since it is normally abundant under both physiological and pathological con-  panied by increased CRP and proteinuria levels in kidney transplant recipients, thus indicating its inflammatory role. 41Although YKL-40 showed many important benefits, the pathophysiological mechanism that leads to its expression in cases of AKI remains uncertain and validated cutoffs remain largely absent.

CONCLUSION
The results regarding the Klotho, CYR61 and YKL-40 biomarkers showed markedly equivocal performance in the previous studies and did not fulfill the expectations that these factors would form valid possible biomarkers for AKI.
Furthermore, Xu et al. conducted experiments on renal cell lines under conditions of hypoxia and found that CYR61 expression prevented apoptosis through phosphorylation of BAD, which released anti-apoptotic factors (bcl-2, bcl-xl) and enhanced cell proliferation through activation of the

ditions; 2 )
its rapid decline over time in spite of AKI progression; 3) the insensitivity of the immunoblotting technique used in quantification in urine; and 4) the fact that most studies were conducted on animal models because of difficulty in obtaining samples from human patients without prolonged routine registry for clinical trials in accordance with the World Health Organization (WHO) requirements and without prior patient approval.Hall et al. showed that increased levels of urinary YKL-40 of up to 5 ng/ml were moderately correlated with AKI progression and/or mortality among patients.Moreover, apparent increases in YKL-40 levels in urine were observed in cases of kidney transplantation among patients hospitalized within 24 hours of PCR = polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay.

Table 2 .
Outlines of the search strategies used for each database 29 the other hand, Seo et al. studied human subjects and showed that renal Klotho levels were reduced, compared with high serum creatinine levels, according to AKI severity.49Likewise,Castellanoetal. observed that Klotho levels were sigwere lower in AKI patients than in healthy individuals and recommended that this should be a candidate biomarker for AKI.29Surprisingly, Torregrosa et al. concluded that there was no difference in urinary Klotho levels measured by means of the ELISA (enzyme-linked immunosorbent assay) technique between AKI

Table 3 .
Summary of characteristics and main results of the 17 previous studies included in this review Recently, Liu et al. showed that there was a notable immediate decline in serum Klotho levels in AKI patients compared with non-AKI (101 ± 16.93 versus 121.64 ± 19.87) after cardiac valve replacement surgery, although the preoperative levels had been steady and close together without any significant difference.Subsequently, 24 hours after the operation, the levels exhibited stepwise recovery towards the preoperative (baseline) levels.This observation indicated that serum Klotho might be a sensitive biomarker limited to a short time after sur-

Table 4 .
36,38iption of biomarkers, their functions and measurement methods AKI.40Further proof was presented byMaddens et   al.,showing that urinary levels of YKL-40 were elevated in septic AKI patients.Taken together, YKL-40 with the best renal troponins (NGAL) might improve stratification of the risk of AKI among patients without any indications of primary renal damage and strengthen early prediction of sepsis-induced AKI.36,38Another study bySchmidtand Malyszko et al. reported that urinary YKL-40 was better than serum YKL-40 levels for distinguishing between delayed graft function and slow or immediate graft function, within 3 days after kidney transplantation.Delayed graft function produces greater severity of ischemic kidney injury, while the damage from other types tends to become repaired. 39,42Synergistically, Hall et al. recommended that urinary YKL-40 could be used as an accurate and reliable biomarker to identify patients at risk of AKI following transplantation, rather than urinary or plasma NGAL. 40Conversely, a pilot study by De Loor et al. demonstrated that the urinary concentrations of YKL-40 and NGAL in ICU patients with AKI stage ≥ 2 measured within 12 h or 24 h exhibited higher convergent diagnostic performance than did serum YKL-40, which did not show any predictive power against AKI. 37Moreover, Tatar et al. concluded that high levels of serum YKL-40 was accom- developing