The Influence of Regular Hemodialysis on the Highly Sensitive Troponin-I Level in Children without Any Symptoms

Backgrounds: Cardiovascular diseases are still the prominent cause of death in cases of end-stage renal disease, cardiac troponin I (cTnI) can be used for detecting cardiac involvement in asymptomatic cases of end-stage renal disease on hemodialysis. Aim: Determine the direct cardiac consequence of dialysis treatments in children on hemodialysis by measuring high-sensitive troponin-I as a marker of myocardial injury. Subjects and Methods: This case-control study included thirty children with end-stage renal disease on regular hemodialysis; the study group was selected from the nephrology hemodialysis unit of Al-Zahraa Hospital, Al-Azhar University. Another group of thirty healthy children matches age and sex with the patient’s group as a control. Highly Sensitive cTnI (hsTnI) was measured pre and post hemodialysis with a sensitive assay; moreover, ECG, lipid profile including cholesterol, triglyceride, low and high-density lipoprotein (HDL) in the same line with routine investigations for those patients, we used bioimpedance for dry weight assessment in the hemodialysis (HD) group. Results: Children on (HD) have a significantly higher (hsTnI) pre-dialysis (0.250 ± 0.069 ng/ml) compared to post-dialysis (


Introduction
Cardiovascular disease (CVD) is the most common cause of death in pediatric CKD patients [1] [2] [3]. For children on regular hemodialysis, the mortality associated with cardiac disease is one thousand times higher than in normal children [4]. CVD was the leading cause of mortality in patients undergoing dialysis, affecting 33% of cases in a cohort of US children, followed from 1995 to 2010 [5].
Children and uremia patients with uremia have a similar constellation of ischemia-predisposing factors to adults, including vascular calcification, increased intima-media thickness and pulse wave velocity, early atherosclerosis, and endothelial dysfunction [6] [7], but without significant atheromatous coronary artery disease.
The incidence of HD-induced hemodynamic disturbance is comparable to adults, with a 20% to 30% incidence of intradialytic hypotension associated with a relative blood volume reduction of 20% to 25% [8].
High-sensitive troponin I (hsTnI) and high-sensitive troponin T (hsTnT) are markers of cardiac damage. Cardiomyocyte necrosis increases its blood levels. It is known that dialysis is cardiotoxic, resulting in a lack of contractility of certain myocardial segments. This mechanism is primarily due to hypoperfusion of the myocardium during dialysis.
The dialysis itself increases cardiovascular risk in patients by many different mechanisms. It has been proven that the incidence of heart failure is much more frequent in patients on hemodialysis than in healthy populations [9]. Studies regarding the effect of HD on TnI levels, measured by conventional assays, are contradictory [10] [11].
Few studies were using hsTnI assay in HD patients. The percentage of asymptomatic HD patients who had pre-dialysis hsTnI levels higher than the reference cutoff point was 5% -51%. The studies that used the newer hsTnI assay still provided limited data [12] [13].
The most common conditions that lead to elevation of the troponin level in the blood are myocardial infarction, toxic damage to the heart, and pulmonary embolism [14].
Children are the best model of patients with uremia for defining the risk for

Subjects and Methods
This case-control study was carried on 30 children with CKD on regular HD and 30 children as a control group matched age and sex with patients group. They were selected from the outpatient clinic and HD unit of the Al-Azhar University Hospital. The patient group was on regular HD for longer than three months at the time of the study [15], for four h/setting, three times weekly, using low-flux polysulfone dialyzer and 4008 Fresenius machine. The most common cause of CKD in the patient's group was acquired 11 (35.0%), congenital causes 8 (26.7%) and hereditary causes 4 (13.3%), and unknown causes in 7 (23.3%). Patients with congenital or acquired heart disease, heart failure, or any other chronic illness excluded from the study. Informed consent was obtained from the parents of the participating children in adherence with the guidelines of the Ethical Committee of Al-Zahra Hospital, Al-Azhar University, Cairo, Egypt. Sample collection and laboratory investigations of children pre-HD: Under a complete aseptic condition, a volume of 10 ml of venous blood was withdrawn from each subject after fasting for 12 hrs. We divided the blood sample into two aliquots; the first aliquot of 2 ml of blood was transferred into an EDTA tube for measuring complete blood count (Sysmex XK-21, Japan). 2nd aliquot of 8 ml was transferred into two serum gel separator tubes and centrifuged for separation of serum. We divided the serum into four parts. The 1st part of the serum was used for the measurement of urea, creatinine, and lipid profile (total cholesterol (CHO), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), total blood Ca, and phosphorus (Ph+) using chemistry auto-analyzer device (Cobas Integra 400 plus, Roche diagnostics, Germany). The 2nd part was used for the measurement of electrolytes, including sodium (Na+) and potassium (K+), by electrolyte analyzer (AVL, 9180 Roche diagnostics, Germany). We used the 3rd portion to measure serum ferritin and PTH by immunoassay using direct chemiluminescent technology (Cobas e411, Roche diagnostics, Germany by chemiluminescence technique). The last portion was stored at −20˚ to measure cardiac-specific Troponin I (cTn-I), which was done by enzyme-linked immunosorbent assay (ELISA).
Another sample, about 2 ml blood, was withdrawn after dialysis and transferred to serum gel separator tube and centrifuged again for measurement of (hsTnI) post-dialysis for comparison with its value before dialysis, which (ELISA also measured).
ELISA assay for measurement of (cTn-I): ELISA using kits supplied from Beta trade, Diagnostics, USA, with Lot NO319081302 Rev. K according to Manufacturer instructions with a lower detection limit of 0.010 ng/ml and sensitivity of 0.04 ng/ml. Elisa system used was varioscan lux, thermo-scientific; USA (well washer).
Resting ECG: Resting ECG (monitors Ltd., Rostov-on-Don, Russia) for the Open Journal of Nephrology patient's group after 5 minutes of complete flat rest pre-hemodialysis, 12 standard leads for the hemodialysis group.
Statistical analysis: Data were collected, revised, coded, and entered into the Statistical Package for Social Science (IBM SPSS) version 23. Spearman correlation coefficients to assess the correlation between two studied parameters in the same group. Receiver Operating Characteristic (ROC) curve was used to assess the best cutoff point with sensitivity and specificity. Interpretation of probability values was as follows: p > 0.05: non-significant, p < 0.05: significant. Table 1 shows a significant decrease in weight and height in hemodialysis children than healthy controls; meanwhile, there is a significant increase in the systolic, diastolic blood pressure, and heart rate compared to healthy controls. Table 2 shows a significant decrease in Hb, platelet counts, and HDL serum level; meanwhile, there is a significant increase in serum urea, creatinine, phosphate, and triglyceride in hemodialysis children than in their controls. Table 3 shows a significant increase in serum level of hsTnI in children pre-HD than healthy controls, but it significantly reduced in post-dialysis reached to be of no significant difference compared to healthy controls. Table 4 shows ECG abnormalities in the patient's group before the hemodialysis; despite the study, patients with no cardiac symptoms, prolonged QT, and QTC were detected in 16 (53.3%) and 10 (33.3%), respectively. Meanwhile, inverted T and LV enlargement were detected in 7 (23.3%) and 6 (20.0%), respectively.     Table 5 shows a significant positive correlation between troponin with systolic and diastolic blood pressure, heart rate, serum urea, creatinine, phosphate, ferritin, PTH, prolonged QT and QTC, but there is a significant negative correlation with Hb, Htc%, and HDL serum level.   Figure 3 show that the specificity and sensitivity of hs-cTnI in early detection of early myocardial injury pre HD is 96.67% and 90.1%, respectively.

Discussion
Established high prevalence of cardiovascular mortality in patients on hemodialysis without current or ongoing cardiac events. Troponin (Tn) is a component of a heart muscle, and its release indicates early events in heart tissue degeneration, necrosis, and myocyte damage [16]. Cardiac troponins (cTnI) are sensitive markers of myocardial injury and play an essential role in diagnosing cardiac ischemia [17]. There is scarce data on the significance of cTnI levels in hemodialysis patients [18]. No studies were using hsTnI assay in HD children, and almost all were in the adult population.     In the current study (cTnI) measured by sensitive assay pre-HD and repeated after hemodialysis in children without cardiac symptoms, we found a significantly high (cTnI) pre-HD. Cardiac troponin-I values are generally not elevated in children with stable cardiac disease or general pediatric conditions [19].
Asymptomatic children with higher pre-dialysis hsTnI levels than the reference cutoff point was (73.30%); troponin is challenging to detect in unaffected muscle, but troponin levels rise several hours after the onset of myocardial injury [16] [28]; we found that a more extraordinary rise of cTnI pre-dialysis was significantly associated hypertension, anemia, elevated urea, and creatinine. Some cross-sectional studies on clinically stable HD patients have found hscTnI is strongly correlated to left ventricular dysfunction and hs-cTnT to coronary artery disease based on a single troponin value [29]. Other conditions that may lead to troponin release are subclinical ischemic heart disease, anemia, arrhythmias, hypertension, angina [30], physical exertion [31], myocardial stunning [32], and intradialytic hypotension [33]. Troponin is released during myocardial damage and due to the loss of myocyte contraction force. After starting onset of irreversible cardiomyocyte damage occurred a similar release of intact cTnI and cTnT and their degradation products due to cardiomyocytes' metabolic inhibition [34] [35]. Increased troponin levels serve as a sensitive and specific biomarker of myocardial injury during the early stages and refer to the beginning of a micro infarct [36]. However, no morphological finding was reported showing the damage in the echocardiography [37]. We recorded in the present work cTnT emerges as a sensitive and specific fore detection of silent myocardial injury in hemodialysis children.
Their study on the association between anemia and T troponin reported a relationship between increased troponin and inadequate erythropoiesis and decreased hemoglobin and considered it a reflection of cardio-myopathy injury [38].
Growth failure and protein-energy wasting may be related to higher troponin levels; we observed it in the current study patients group and typical for the uraemic phenotype [44]. Elevated troponin could be explained because PEW is related to fluid overload in dialysis patients [45]. The dry weight towards which a patient's dialysis prescription is complex and falsely determined in PEW's presence leads to chronic fluid overload, myocardial stretch, and troponin release.
In the current study, the HDL fraction and triglyceride are significantly elevated in hemodialysis children, and HDL fraction had an inverse relationship with hs-cTnI. This result is consistent with de Goma et al. (2001) [46], where an inverse relationship between HDL cholesterol and the prevalence of coronary heart disease (CHD) was observed. This relationship is probably due to HDL's role in transporting cholesterol from peripheral tissue to the liver for its subsequent catabolism and excretion Nayak et al. (2010) [47].
In our study, the patients' electrocardiography was also recorded and inter-Open Journal of Nephrology preted by the cardiologists in the current study. We observed prolonged QT and QTC of ECG patterns, and this pattern is associated with high troponin levels in hemodialysis children. The prevalence of acquired long QT syndrome is high and increases with kidney function decline in CKD patients [48]- [55]. Also, the risk of QTc prolongation and the inverted T wave is higher in a hemodialysis patient. Hemodialysis patients had a series of poor conditions, such as volume overload, metabolism disorder, and uremic toxin accumulation, which lead to asymptomatic myocardial damage [56], which strengthens the current study finding also Ozdemir et al. (2005) [57] had reported greater QTc interval compared to control subjects and concluded that children receiving hemodialysis might be at greater risk of ventricular arrhythmia and sudden death. In contradiction to Valsangiacomo et al. (2007) [58], conducted on nine children, no changes in the QTc related to hemodialysis. This discrepancy may reflect the smaller number of cases included in their work.
Also, increased QT interval and dispersal on an electrocardiogram (ECG) are associated with LVH; and is detected in 6 (20.0%) of the current study cases; LVH is an adaptive response to chronic pressure and volume overload (allowing maintenance of systolic function) and is the most common cardiovascular abnormality in children with CKD, Nashwa et al. (2009) [59]. Beaubien et al. (2002) [60] reported similar findings.
The current study showed a significant relationship between hs-cTnI and ferritin; there is no previous study recorded this finding in hemodialysis children, but Shahramian et al. (2013) [61] reported in micro infarct, troponin increases independent of ferritin, but this finding in patients with thalassemia and iron overload without cardiac symptoms. Also, Wood (2011) [62] studied the impact of iron assessment by MRI and stated that an increase of ferritin would increase the risk of cardiac toxicity.
In the present work, there is a significant association between hs-cTnI with phosphate and PTH serum level; the hypothesis shows the deleterious effect of high phosphate and PTH on the cardiovascular system and is reflected in the current investigations by elevated hsTnI. Van Ballegooijen et al. (2013) [63] reported a significant similar association between PTH receptors have been demonstrated in the heart and exert a trophic effect on cardiomyocytes; furthermore, PTH activates protein kinase C, which could lead to hypertrophic growth and expression of fetal type proteins in cardiomyocytes [64]. Moreover, it might contribute to biochemical changes and an increase in LV mass and incident heart failure [65] [66] [67].
In the current study, significant low platelets count in hemodialysis children, aggregation of platelets during dialysis may be due to exposure of blood to the roller pump segment of the dialysis tubing or microbubbles. Heparin used in hemodialysis may also contribute to HD-associated platelet activation and thrombocytopenia [68] [69]. Platelets secrete and express many crucial mediators of coagulation, inflammation, thrombosis, and atherosclerosis [70] [71]. A significant association of blood platelet counts and troponin was detected; these find-Open Journal of Nephrology ings raise the hypothesis of platelets' potential importance in the underlying pathophysiology of cardiovascular disease.
In conclusion, children on regular hemodialysis might have silent myocardial ischemia detected by hs-cTnI before hemodialysis with multifactorial etiologies; the nephrology team should aggressively treat those patients to prevent further myocardial damage, and hsTnI emerges as a highly sensitive and specific marker for early diagnosis of early myocardial damage in hemodialysis children.