Study design and population

This cross-sectional study was conducted between November 2022 to March 2023 at the HD Center of An-Najah National University Hospital (NNUH), Palestine. NNUH, a teaching tertiary referral hospital has the largest dialysis center in the area. Eighty-one HD patients participated in this study. We included patients who were over the age of 18 and had been on HD for more than six months. Those with recent onset (less than two weeks) of symptoms of the acute coronary syndrome (chest pain radiating to jaw, neck, shoulders, or left arm) or with a known history of chronic inflammatory disease (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, and Crohn’s disease), or those with underlying infection, and who are not reliable due to medical or psychological conditions were excluded from the study.

We calculated the sample size using the EpiInfo software’s "Sample Size for Comparing Two Means" method, where the predicted h-cTnT in the IL6 high group was 57±10.2 and in the IL6 low group was 50±10.4. The confidence interval (2-sided) was 95%, and the power was 80%. This resulted in a sample size of 76. Participants were selected conveniently; 81 patients agreed to participate and met the criteria.

Variables and measurement tool

Demographic and clinical data were collected from the participant’s medical records. This includes age, gender, body mass index (BMI), duration of HD, type of vascular access, smoking habit, chronic respiratory disease, history of malignancies, diabetes, hypertension, previous history of ischemic heart disease (IHD), cerebrovascular accident (CVA), and peripheral arterial disease (PAD). Patients were divided into groups according to the presence or absence of these comorbidities. We reviewed previous patients’ medical records, looking for patients who underwent percutaneous cardiac catheterization to investigate if they had ectatic and \ or stenotic vessels.

Two blood samples were drawn in two tubes (EDTA tube and lithium heparin tube) for 81 patients. Samples were assayed for IL-6 and h-cTnT levels. Blood samples were centrifuged and stored at -20 c for one week until the samples were collected. The h-cTnT was analyzed by fifth-generation electroluminescence immunoassay using Cobas e 602(Roche Diagnostics). The detection limit was three ng/L, and the coefficients of variation below 10% were 11 ng/L. The cutoff at the 99th percentile was 14 ng/L.

Plasma IL-6 levels were measured using Diaclone human IL-6 enzyme-linked immunosorbent assay (ELISA) set. The sensitivity or minimum detectable limit for IL-6 was 2pg/mL, and the intraassay coefficient of variation was 3.6%. The normal value of IL-6 is below 7 pg/ml. Patients with high troponin levels above the upper reference limit of the average population (14ng/L) were assessed clinically if they had any signs or symptoms of acute coronary syndrome. Additionally, serum levels of albumin, calcium, iron, phosphate, potassium, sodium, hemoglobin, platelet count, WBC count, parathyroid hormone, and bicarbonate were measured at An-Najah National Hospital Laboratory with methods used in clinical routine.

Statistical analysis

Statistical Package for Social Sciences (SPSS) version 28 software was used for data entry and analysis. Descriptive analysis was used to describe the characteristics of participants using frequencies and percentages for categorical variables, mean ± standard deviation (SD) or medians, and interquartile ranges for continuous data. The normality of the data was checked using the Kolmogorov—Smirnov test. The h-cTnT and IL-6 levels were not normally distributed; therefore, medians and interquartile ranges were reported, and non-parametric analysis was employed. We used the Mann-Whitney U and Kruskal-Wallis tests to examine whether demographic and clinical variables are associated with h-cTnT and IL-6. Spearman correlation coefficient was used to test the correlation between h-cTnT, IL-6, and other continuous variables. This study had no missing data and a P-value of less than 0.05 was accepted as significant.

The research received approval from the Institutional Review Board (IRB) at An-Najah University, with reference number Med. June 2022/8. Patients were invited to participate in the study voluntarily, with a thorough explanation of the study’s aims, objectives, and potential risks. Individuals who agreed to participate signed a written informed consent. No personally identifiable information was gathered from participants, who were referred to using unique codes to ensure confidentiality. Access to the data collected was tightly controlled and limited to the research team for the sole purpose of conducting the study.

Background characteristics

A total of 81 HD patients were included in the study. Median age was 58 (IQR = 20–83) years, with 49 (60.5%) of these patients being male. The median duration of dialysis was 48 (IQR = 7–240) months, and 70 (86.4%) patients had fistula, whereas others had catheters. The clinical characteristics of these patients are summarized in (Table 1).

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Table 1. Sociodemographic and clinical characteristics of the participants.

https://doi.org/10.1371/journal.pone.0296965.t001

H-cTnT and IL-6

The median values of h-cTnT and IL-6 were 62.8 ng/L (IQR = 7.6–255.6) and 2.1 pg/ml (IQR = 0.1–64.8), respectively (Table 2). Only one patient (1.2%) had h-cTnT below the upper reference limit for the average of normal population, which is it less than 14 ng/L. On the other hand, two patients (2.5%) had IL-6 above 43.5 pg/ml, the highest value detected recently in the average population, whereas the normal IL-6 range is below 7 pg/ml.

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Table 2. Distribution of Intrlukin-6 and Troponin levels among HD patients.

https://doi.org/10.1371/journal.pone.0296965.t002

H-cTnT and IL-6 values were significantly correlated (rho = 0.463, P<0.001), as were h-cTnT level with age (rho = 0.519, P<0.001), albumin (rho = -0.297, p = 0.007) and transferrin saturation (rho = -0.227, P = 0.042). Furthermore, IL-6 level was correlated with age (rho = 0.422, P<0.001), albumin (rho = -0.389, P<0.001), iron (rho = -0.382, P<0.001), and transferrin saturation (rho = -0.362, P = 0.001) (Table 3).

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Table 3. Correlation between h-cTnT, IL-6, background, and lab characteristics.

https://doi.org/10.1371/journal.pone.0296965.t003

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Fig 1. Linear regression analysis of IL-6 level in relation to h-cTnT level.

https://doi.org/10.1371/journal.pone.0296965.g001

Patients with diabetes and hypertension had significantly higher median h-cTnT levels (70.8 ng/L; P = 0.003) and (65.1ng/L; P = 0.027), respectively, as compared to non-diabetics and patients without hypertension (Fig 2 and Table 4).

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Fig 2. h-cTnT level distribution in groups divided according to DM and HTN.

https://doi.org/10.1371/journal.pone.0296965.g002

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Table 4. Patient’s background and clinical characteristics with h-cTnT and Interleukin 6.

https://doi.org/10.1371/journal.pone.0296965.t004

Patients with a history of IHD and CVA showed significantly higher median h-cTnT levels (75.5 ng/L; P = 0.001), (70.9 ng/L; P = 0.013), respectively, than those without a history of these conditions. Moreover, males exhibited significantly higher median h-cTnT levels (73.9 ng/L; P <0.001) than females. On the other hand, the median IL-6 level did not differ significantly between groups based on gender, BMI, smoking status, and other comorbidities. In addition, there is no difference in the median values of h-cTnT and IL-6 among patients classified by vascular access type (Table 4).

Discussion

This study demonstrated that h-cTnT and IL-6 are significantly correlated, suggesting an association between ongoing inflammation and h-cTnT concentrations in HD patients without acute symptoms of myocardial ischemia. This finding is consistent with older studies showing a correlation between h-cTnT and other inflammatory markers like CRP in HD patients [23]. On the other hand, Chen et al. showed in a cohort study of 575 dialysis patients that there was no significant association between h‐cTnT and CRP level [24]. In non-dialysis patients with acute myocardial ischemia, h-cTnT and IL-6 levels are raised and used to predict further cardiac events [25,26]. Atherosclerosis is an inflammatory disease that causes extracellular matrix remodeling, oxidative stress, endothelial dysfunction, inflammation in the arterial wall, and progressive lipid accumulation [13]. It can cause multiple clinically significant CVDs, including IHD. Following myocardial ischemia, cardiomyocytes would experience irreversible necrosis from hypoxia, followed by the infiltration of inflammatory cells and the release of pro-inflammatory factors like IL-6. Moreover, cardiomyocytes produce IL-6 under hypoxia and ischemic stress [25], which could explain the association between inflammatory markers and h-cTnT. Although this study is not powered to look at the incidence of cardiomyopathy and coronary atherosclerosis in the group of patients with elevated level of h-TnT and IL-6, particularly that those variables were found to elevated in diabetics and hypertensives who are at higher risk of having cardiomyopathy and coronary atherosclerosis [27,28]. It would be interesting to look at this possibility in future research by adding echocardiography and either invasive or non-invasive test to look for coronary atherosclerosis.

The correlations with other negative acute phase reactants like transferrin saturation and serum albumin further underscored the association between h-cTnT and inflammation in the current study. As expected, we also found that IL6, a potent inflammatory marker, has an inverse correlation with serum iron level, transferrin saturation, and albumin level. Inflammation affects intestinal iron absorption and sequester iron in macrophages, decreasing serum iron levels and transferrin saturation [29]. Ferritin is also a marker of acute and chronic inflammation. Patients with CKD had hyperferritinemia due to iron overload and chronic inflammation [30].

In contrast to a previous study that showed a positive correlation between ferritin and inflammatory markers like IL-6 and CRP in patients with HD [31], we found that ferritin has an inverse correlation with h-cTnT and IL-6. However, our study’s results were not statistically significant, which could be attributed to the small sample size. Malnutrition may cause a drop in serum albumin level, although inflammation can cause this as albumin is a negative acute phase reactant [32]. Thus, HD patients with the lowest albumin concentrations are those with both vascular disease and inflammation. In addition, previous studies demonstrated that hypoalbuminemia is one of the strongest predictors of mortality in ESRD [33]. As we discovered a strong association between inflammatory markers and h-cTnT, the present question is whether the use of drugs with anti-inflammatory effects will help in decreasing CVD mortality in asymptomatic HD patients.

The minimum and maximum levels of h-cTnT in HD patients without acute coronary syndrome were also assessed in this study; the h-cTnT range was 7.6 ng/L to 255.6 ng/L. Even higher h-cTnT level (738.30 ng/L) has been reported in HD patients without acute ischemia [34]. As was previously mentioned, even in the absence of acute episodes of myocardial ischemia, blood troponin concentrations in ESRD patients tend to be greater than in the general population [35]. We also discovered many patients with elevated h-cTnT (98.8%), similar to other studies on h-cTnT [36,37]. Although the pathophysiological basis and the biochemical processes underlying the elevated h-cTnT concentrations in ESRD are still unclear, it is almost attributed to chronic myocardial damage rather than acute myocardial infarction as these patients were clinically stable and did not have any signs or symptoms of acute ischemia [34]. Estimated glomerular filtration rates affect the metabolism and clearance of h-cTnT, so this could explain the elevation of troponin in CKD [38]. Volume removal and overload may play a role in inducing ischemia and elevation in troponin. Rapid changes in blood pressure during dialysis treatment are considered a risk factor for IHD. Intradialytic ultrafiltration causes hypotension and a decrease in venous return with a subsequent increase in the stretching of the heart without compensation, particularly in patients with chronic dialysis and heart failure, which leads to myocardial ischemia due to hypoperfusion [39], thus higher troponin levels.

Several hypotheses have been proposed about the elevation of cardiac troponins in HD patients that suggest myocardial injury. Left ventricular hypertrophy was a factor highly predictive of high serum levels of cardiac markers [40]. Some cross-sectional studies on clinically stable patients with ESRD have found that hs-cTnI was strongly correlated to left ventricular dysfunction and h-cTnT was correlated to coronary artery disease based on a single troponin value [41]. Rather than coronary artery stenosis, microvascular lesions or direct injury to myocardial cells (e.g., toxic, stretching, hypoxia, apoptosis) encountered in the cardiomyopathy of HD patients was associated with high cardiac troponins. The co-existence of heart failure, even without acute ischemia, non-ischemic clinical conditions, including tachy/bradyarrhythmias and aortic valve disease, were all associated with high cardiac troponins [40]. Another study found that high cardiac troponin level was associated with increased risk for all-cause and CVD mortality, as mortality risk was doubled in patients with persistently high levels of h-cTnT in ESRD [42]. However, what remains to be established is whether a higher reference limit of cardiac troponins will be used to diagnose acute coronary syndrome in patients with ESRD and if these patients with acute elevations in troponins are accurately identified as those who would benefit from early coronary interventions, such as administration of IIb/IIIa anti-platelet inhibitors or coronary revascularization, as was confirmed in the general population. Further studies are needed to investigate whether more specific diagnostic tests, such as coronary angiography, would lead to interventions that improve the outcomes in patients with chronic elevations of troponins and inflammatory markers.

Diabetes, hypertension, previous history of coronary artery disease, and cerebrovascular accident were found in this study to be associated with elevated concentrations of h-cTnT, which have been reported by other studies [24,43,44]. Findings of higher serum h-cTnT in diabetic HD patients are consistent with previous data from HD patients reported by others [36]. Advanced glycosylation end-products could alter membrane integrity and promote h-cTnT expression in noncardiac cells, which could account for this elevation. Chronic hyperglycemia also affects heart microcirculation, which results in microvascular damage and, in turn, ischemia, both of which raise troponin levels [45]. Šimić et al. suggested using other cardiac damage markers, such as copeptin and heart fatty acid binding protein, which have different elimination mechanisms [45]. Moreover, diabetic patients are more likely to develop microangiopathy and myocardial ischemia, which may be clinically silent and undiagnosed in these patients. We also found a significant association between h-cTnT level and hypertension in the current investigation, as chronic hypertension is associated with arterial wall stiffness and atherosclerosis.

Another key finding in our study is that age and male gender correlated significantly with the h-cTnT level of the HD population, supporting earlier reports [35]. This study discovered that age and the male gender are both independently related to the risk of increasing h-cTnT levels. While it seems that local inflammation, microvascular damage, and atherosclerosis are connected with older age, the explanation of male gender is less obvious. Male gender is associated with a higher left ventricular mass index [46], but in another study, these associations persisted after adjustment for the left ventricular mass index [35]. In our study, neither the duration of HD nor the type of vascular access was associated with h-cTnT levels.

This study has possible limitations. First, the cross-sectional study design limits the ability to suggest causality between related variables. Second, the results should be cautiously generalized because the study was conducted at a single dialysis unit with a small sample size. Thirdly, not all patients were evaluated with echocardiography, so elevated cardiac troponin could be associated with other causes like left ventricular hypertrophy rather than atherosclerosis. Since cardiac catheterization and ECG were not used to investigate patients with high cardiac troponin levels, it is impossible to rule out the possibility of an acute cardiac event in these asymptomatic patients.