Cystatin C as a biomarker of acute kidney injury in a group of critically ill children in a pediatric intensive care unit

1Department of Pediatrics, Mashhad University of Medical Sciences, Mashhad, Iran 2Clinical Research Unit, Mashhad University of Medical Sciences, Mashhad, Iran 3Department of Community Medicine, Mashhad Branch, Islamic Azad University, Mashhad, Iran 4Department of Internal Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 5Clinical Toxicology Ward of Imam Reza Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran


Introduction
Acute kidney injury (AKI) is a condition that is frequently seen in critically ill children in the pediatric intensive care units (PICUs) and is associated with poor clinical outcomes (1,2).It is defined as a sudden cessation of kidney function and is sometimes associated with catastrophic life-threatening consequences in children admitted to PICUs (3).Critically ill pediatric patients are prone to this condition and its incidence has been reported between 18%-52% in such patients (4).RIFLE (Risk, Injury, Failure, Loss, and End-stage kidney disease) and AKIN (Acute Kidney Injury Network) criteria have been proposed to DOI: 10.15171/jrip.2018.58diagnose AKI (5)(6)(7).In 2012 these two classifications were combined in the KDIGO guideline for diagnosing AKI (8).Serum creatinine is used to detect and to stage AKI in these guidelines although its limitations are well-known.Serum creatinine can be influenced by non-renal factors such as muscle mass, liver function and dietary creatinine intake, medications, gender and age.Rise in serum creatinine may be delayed for days after renal injury.According to these limitations, researchers have proposed a number of novel biomarkers for the early detection of AKI in recent years (9,10).One of these biomarkers is serum cystatin C which seems to be a useful marker for the early detection of AKI (11).Numerous studies have shown that serum cystatin C acts as a good glomerular filtration rate (GFR) marker (12)(13)(14).Cystatin C is a proteinase inhibitor that is constantly produced by nucleated cells in the body and is freely filtered by the glomerulus and is entirely reabsorbed in the proximal tubule (15,16).Recent investigations have proposed that serum cystatin C is a better marker of AKI (15,(17)(18)(19).

Objectives
We conducted this study to compare between serum creatinine and serum cystatin C in identifying critically ill children with AKI.

Study population
This was a prospective study.It was conducted in PICU of Dr Sheikh's pediatric hospital in 2014.The hospital is affiliated to Mashhad University of Medical Sciences and is the only pediatric tertiary care hospital in east of Iran.All critically ill children were older than 6 months when admitted PICU.Exclusion criteria were: age younger than 6 months, those with chronic kidney problems and patients whose parents were not willing to take part in the study.

Laboratory assessments
Serum cystatin C (using human cystatin C ELISA kit) and creatinine (using Jaffe colorimetric method) were measured on admission to PICU.They were rechecked every 48 hours for 3 times.Then each serum cystatin C and creatinine measurement was compared with GFR of patients to see which marker was able to show the onset of AKI earlier?
Ethical issues 1) The research followed the tenets of the Declaration of Helsinki; 2) informed consent was obtained from the parents of children and 3) the research was approved by the ethical committee of faculty of medicine of Mashhad University of Medical Sciences (Grant #910431).

Data analysis
All data was coded and analyzed using SPSS 21.Descriptive studies were used for the calculation of mean and standard deviations (SDs).Chi-square test was used for association between parameters.We also used ROC curves for comparing different measured predictors and to assess sensitivity and specificity levels and P value below 0.05 was assumed to be significant (P < 0.05).

Results
Fifty-four critically ill children older than 6 months who were admitted in the PICU, entered the study.Mean age of patients was 4.09 ± 3.98 years (minimum; 6 months and maximum was 15 years old).Male gender was predominant (70.4%, n = 38).Mean height was 91.8 ± 23.3 cm and mean weight was 13.15 ± 7.29 kg.Demographic data of patients are shown in Table 1.Around 26 patients did not survive and six of them died during the first 3 days of PICU admission, which were excluded from the study.The other 20 patients, died after the fifth day (last sampling for creatinine and cystatin C).On fifth day of the study, 38 patients (70.3%) experienced AKI according to RIFLE criteria (risk 43.7%, injury 27% and failure 8.3%).Table 2 shows changes in creatinine and cystatin C over time.Analysis of variance (ANOVA) shows that cystatin C had a significant change over time in all patients.Additionally cystatin C elevation had a significant correlation with the stage of AKI (P < 0.001).Alterations of cystatin C were most prominent in the patients at the failure stage of AKI.ROC analysis was performed at each time point to evaluate the usefulness of serum cystatin C for the diagnosis of AKI (Figures 1-3).In the first day, the AUC for cystatin C and creatinine were 0.93 (P = 0.005) and 0.67 (P = 0.268) respectively.The diagram shows that cystatin C had a greater AUC and thus was better able to identify AKI.
In the third day the AUC for cystatin and creatinine were 0.96 (P = 0.003) and 0.95 (P = 0.004) respectively.These findings showed that cystatin C had a greater AUC and therefore had better ability to identify AKI.However AUC of creatinine was very close to that of cystatin C and there was no significant difference between these two entity for diagnosis of AKI (P > 0.05).
In fifth day, the AUC for both creatinine and cystatin C was 0.96 and thus these two test had the same ability for the diagnosis of AKI at this time point.

Discussion
In our study 70.3% of the critically ill children experienced AKI which is greater than the reported incidence of AKI in PICU patients (1,20).The greater proportion may be due to the fact that we had a limited number of PICU beds in our center, thus very ill children were admitted in the PICU.This great proportion shows that AKI is an important and prevalent condition in the PICUs and early detection and management of this condition seems mandatory.Currently, serum creatinine is the most commonly used laboratory test for the estimation of kidney function in pediatric patients.However, due to its being influenced by age, gender, muscle mass, diet and administration of drugs, it is not enough sensitive for early detection of changes in GFR (19,21).According of these limitations, numerous studies have focused on new biomarkers for earlier and more reliable detection of AKI (9,10).One such biomarker is cystatin C. In this    study we hypothesized that serum cystatin C would detect AKI in critically ill pediatric patients earlier than serum creatinine.Our results demonstrated that both serum cystatin C and serum creatinine raised in patients with AKI; however, cystatin C increased earlier.Our results were similar to previous studies (22)(23)(24).

Conclusion
In summary serum cystatin C can be used as an early marker of AKI in critically ill pediatric patients.We hope that this study will help to better management of AKI in pediatric patients by institution of early interventions.Also this biomarker may help us identify patients at risk for renal insufficiency like patients with vesicoureteral reflux (25) or those on certain medications with renal complications (26,27) at an earlier stage.Further studies on these issues seem mandatory.

Limitations of the study
Our study had some limitations of course.First of all we used serum creatinine for defining AKI.This study conducted with a small sample and the results should be validated by multi-centric studies with larger sample size.

Figure 2 .
Figure 2. ROC analysis of serum cystatin c and creatinine in the second day.

Figure 3 .
Figure 3. ROC analysis of cystatin C and creatinine in the third day following ICU admission.

Figure 1 .
Figure 1.ROC analysis of serum cystatin C and creatinine in the first day.

Table 1 .
Demographic data of patients with AKI

Table 2 .
Creatinine and cystatin C levels in patients at different stages of AKI ' contribution YR and AF; study design, preparation of manuscript, and final revision.ME; study design, manuscript edition, and final revision.SB and SR; data gathering, data interpretation, and manuscript preparation.YR and AA; data interpretation, manuscript preparation, and final revision.YR; data gathering and manuscript preparation.