|Year : 2015 | Volume
| Issue : 3 | Page : 460-467
|Tumor necrosis factor gene expression in regular hemodialysis patients
Hemmat E El Haddad1, Mohamed A Marie1, Tarek M Samy1, Reem Jan Farid2, Mai Sherif2, Usama A Sharaf El Din1
1 Department of Internal Medicine, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
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|Date of Web Publication||20-May-2015|
| Abstract|| |
This study evaluates tumor necrosis factor (TNF)-alfa gene expression in patients with end-stage renal disease (ESRD) on regular hemodialysis as an expression of cardiovascular disease (CVD) risk even on a sub-clinical level and its relation to some of the parameters incriminated in the pathogenesis and the establishment of uremic arteriopathy. A total of 51 patients with ESRD on regular hemodialysis and 20 healthy subjects matching in age and gender as a control group were recruited. All selected cases were subjected to serum lipid profile, Creactive protein (CRP), TNF-alfa gene expression and Doppler study of carotid arteries to estimate carotid intimal media thickness (cIMT). Serum triglycerides (TGS) level (P <0.001), CRP positivity (P = 0.002), relative quantification (RQ) of TNF-alfa gene expression (P = 0.007) and cIMT (P = 0.02) were significantly higher while high-density lipoprotein (HDL) level (P <0.001) was significantly lower among cases compared with controls. RQ showed a significant positive correlation with CRP titer (rho = 0.583, P = 0.011). Results also showed a significant strong negative correlation between with CRP titer and cIMT (rho = -0.590, P = 0.010). CRP titer showed only a significant strong negative correlation with age (rho = -0.589, P = 0.01) and positive correlation with HDL (rho = 0.51, P = 0.031). Patients with ESRD have increased gene expression of TNF-alfa and CRP titer together with increased atherosclerosis as expressed by increased cIMT.
|How to cite this article:|
El Haddad HE, Marie MA, Samy TM, Farid RJ, Sherif M, Sharaf El Din UA. Tumor necrosis factor gene expression in regular hemodialysis patients. Saudi J Kidney Dis Transpl 2015;26:460-7
|How to cite this URL:|
El Haddad HE, Marie MA, Samy TM, Farid RJ, Sherif M, Sharaf El Din UA. Tumor necrosis factor gene expression in regular hemodialysis patients. Saudi J Kidney Dis Transpl [serial online] 2015 [cited 2021 Jan 16];26:460-7. Available from: https://www.sjkdt.org/text.asp?2015/26/3/460/157309
| Introduction|| |
Cardiovascular disease (CVD) accounts for more than half of deaths among end-stage renal disease (ESRD).  In patients receiving chronic dialysis, coronary artery disease (CAD), especially acute myocardial infarction (MI), is underdiagnosed and under treated despite its high prevalence.  The pathology of CVD observed in patients with ESRD is different from the pathology observed in patients without chronic kidney disease, and is characterized by the presence of both atherosclerosis and arteriosclerosis.  Atherosclerosis is defined as an intimal disease with development of arterial plaques and narrowing of the arterial lumen mainly caused by increased levels of lowdensity lipoprotein (LDL) and vascular inflammation. By contrast, arteriosclerosis is mainly located in the media and results in arterial remodeling with stiff arteries.  An important factor for developing increased calcification in ESRD involves disturbances in the calcium- phosphate homeostasis owing to secondary hyperparathyroidism. 
ESRD is now considered as a typical situation of chronic inflammatory state  and Creactive protein (CRP), a non-specific marker of inflammation, is reported as a fundamental biomarker for cardiovascular risk stratification in these patients.  There is consistent evidence that CRP and pro-inflammatory cytokines such as interleukin (IL-1) beta, IL-6 and tumor necrosis factor-alfa (TNF-α) are risk factors for atherosclerotic complications and predict death and adverse cardiovascular outcomes in these patients. 
Prognostic indicators leading to ESRD in chronic kidney diseases have been studied extensively, of which genetic factors remain a subject of great concern. IL-1 and TNF-alfa are potent pro-inflammatory cytokines that are involved in several chronic kidney diseases. Studies on cytokine gene polymorphism have revealed important information about the role of genetic factors in disease susceptibility and severity. 
Most attempts so far have focused on genes involved in progression of chronic kidney disease (CKD) and in CVD and its underlying complications. These studies have been performed eminently in populations with late stages of CKD. As a large number of CKD patients will die before reaching ESRD, a survivor bias must be acknowledged. Hence, an allele that is more frequently found in the late stages of CKD may in fact be a marker of increased survival. 
TNF gene is located on chromosome 6 in the class III region of the major histocompatibility complex (MHC) and is flanked by the lymphotoxin alfa and beta genes. TNF gene is tightly regulated at the level of transcription. 
Carotid intima-media thickness (cIMT), measured by ultrasonography, is a non-invasive, well-validated tool that has been used as a surrogate for clinical coronary events. cIMT has demonstrable utility as an independent predictor of future cardiovascular events. 
The aim of our study is to evaluate TNF-alfa gene expression in patients with ESRD on regular hemodialysis as an expression of CVD risk even on a sub-clinical level.
| Subjects and Methods|| |
This is a cross-sectional, observational, comparative study that was conducted in the Kasr Al Aini Dialysis Unit, Faculty of Medicine, Cairo University, from January to June 2013. Fifty-one patients with ESRD on regular hemodialysis were included in the study (26 males and 25 females, and their age was ranging from 20 to 70 years) and 20 healthy subjects (ten males and ten females) of matched age and sex were enrolled as controls. All patients provided in-formed consent. All patients were subjected to thorough history taking and physical examination to assess the patients' age, sex, duration of dialysis and any associated medical conditions, with a special emphasis on exclusion of clinical CVD.
Venous blood was withdrawn from all subjects and was divided into two parts: The first part - serum was extracted to assess lipid profile [serum cholesterol, triglyceride and highdensity lipoprotein (HDL) cholesterol and LDL cholesterol] by the Friedewald equation 13 and CRP using a semi-quantitative assay (rapid latex agglutination test) according to the manufacturer's instructions by Omega Diagnostics Limited, Alva FK12 5DQ, Scotland, UK.
TNF-alfa gene expression
The second part of the venous sample was whole blood, which was collected with EDTA anticoagulant. Total RNA, purified from mononuclear cells, was reverse-transcribed and cDNA was amplified by polymerase chain reaction (PCR) primed with specific oligomers in order to determine the TNF-alfa gene expression. Blood samples were processed within a few hours of collection. Total RNA was extracted from all patients and controls using QIAamp® RNA Blood Mini Kits (Qiagen,) Hilden, Germany). The concentration and purity of the extracted total RNA from all patients and controls were measured using NanoQuant from Tecan (Mannedorf, Switzerland). It is the most popular technique for determining nucleic acid concentrations and is based on measuring the absorbance at 260 nm (A 260 ). The purity of the DNA or RNA is checked by comparing absorbance values from the 260 nm and 280 nm measurements (260/280 ratio). The gene expression analysis was performed using a StepOne™ real-time PCR system (Applied Biosystems Waltham, Massachusetts, USA). The principle of gene analysis by this system is using comparative computed tomography experiments (ΔΔCT). Sequence-specific PCR primers and fluorescent probes for mRNA TNF-α were designed using the computer software primer Express Version 1.0 ABI PRISM (Applera Norwalk, Connecticut, USA). 
In all participants, a carotid sonography was performed by means of a high-resolution (ALT 5000 HDI) scanning device, equipped with a phased array 7.5 MHz linear scanner probe and photographed by a SONY video Graphic Printer UP-p890 MD (Panasonic S-VHS, Kadoma, Osaka, Japan). All these exams were carried out by a single specialist physician and all images were photographed. The measurements of cIMT were made about 1 cm proximal to the carotid bulb and always in a plaque-free area. For each patient, three measurements were performed for each side. All results are the mean of the two sides.  With this technique, three parallel echogenic lines separated by an anechoic space can be visualized at levels of the artery wall. It was previously shown that these lines were generated by the blood-intima and media-adventitia interfaces. The distance between the two lines gives a reliable index of the thickness of the intimal-medial complex. Subjects were examined in the supine position. 
| Statistical Analysis|| |
Pre-coded data were statistically analyzed using the Statistical Package of Social Science Software program (SPSS), version 21. Data were summarized using mean, standard deviation (if parametric), median and interquartile range (if non-parametric) for quantitative variables and frequency and percentage for qualitative parameters. Comparison between groups was performed using an independent sample ttest (if parametric) or the Mann-Whitney test (if non-parametric) for quantitative variables and chi-square test or Fisher's exact test for qualitative variables. The Spearman correlation coefficients were calculated to test the association between quantitative variables. Probability (P) values <0.05 were considered statistically significant and <0.01 were considered highly significant.
| Results|| |
Comparison of the demographics and laboratory findings between group 1 (cases of ESRD) and group 2 (normal controls) showed no significant changes between both groups as regards age and sex. It also showed that triglycerides (TGS) level, CRP positivity, relative quantification (RQ) of TNF-alfa gene expression and cIMT were significantly higher among cases than controls, but the HDL level was significantly lower among cases when compared with that among controls. Cholesterol and LDL did not show any significant difference between both groups [Table 1].
|Table 1: Comparison between cases and controls as regards demographics and laboratory findings.|
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Correlations between RQ with other variables within cases only showed that RQ did not show any significant difference as regards sex or CRP positivity, but it showed a significant positive moderate correlation with CRP titer [Figure 1]. RQ did not show any significant correlation with age, lipid profile or cIMT.
Correlations between cIMT with other variables within cases showed that cIMT did not show any significant difference as regards sex or CRP positivity, but it showed a strong, significant negative correlation with CRP titer [Figure 2]. Also, cIMT did not show any significant correlation with age or lipid profile.
Other correlations within cases showed that the CRP titer only showed a significantly strong negative correlation with age [Figure 3] and positive correlation with HDL [Figure 4], but no significant correlation with the other lipid profile parameters.
Relations of lipid profile to CRP positivity among cases only showed that all lipid profile parameters did not show any significant differences as regards CRP positivity [Table 2].
| Discussion|| |
CVD is the leading cause of mortality in uremic patients. In large cross-sectional studies of dialysis patients, traditional cardiovascular risk factors such as hypertension and hypercholesterolemia have been found to have low predictive power, while markers of inflammation and malnutrition are highly correlated with cardiovascular mortality.  IL-6 and TNF-alfa may play important roles in the development of T helper (Th) imbalance, CVD and wasting in the uremic milieu. 
It has been stated by Feldman et al that both in vivo and in vitro studies demonstrated that TNF affects its cellular and biochemical changes in a way that mirror those seen in patients with congestive heart failure.  It is possible that such factors and their genetic variations may not only be involved in the etiology and progression of ESRD but also in the etiology and progression of secondary complications, such as CVD and infections. 
Our study involved the identification of TNFalfa gene expression (as one of the markers of cytokine dysfunction) and CRP (as a nonspecific biomarker of CVD risk) together with the estimation of sub-clinical atherosclerosis by measuring the CIMT in two groups of subjects: Group 1 involved ESRD patients on regular hemodialysis and group 2 involved normal control subjects of matched age and sex.
On comparing both groups as regards demographics and laboratory findings, the results revealed that the relative quantification of the TNF-alfa gene was higher among the group of ESRD than the healthy subjects in group 2. Those results explain the role of inflammatory markers in the pathogenesis of CVD in ESRD, especially the pro-inflammatory cytokines. Studies evaluating the increased level of TNFalfa and its gene polymorphism involving certain pro-motors revealed its incrimination in increasing the cardiovascular risk in ESRD patients. , A study carried out by Chandel et al in 2010 that assessed TNF and IL-6 serum levels and their gene polymorphism in ESRD patients before and after renal transplantation (at the end of the 1st, 3rd and 6th months) revealed that the increased levels of TNF-alfa and IL-6 not only explains their role in increasing CVD risk in ESRD but also their role in graft rejection after renal transplantation. 
The CRP positivity in our study gave a highly significant result on comparing group 1 (ESRD cases) with group 2 (normal controls). Those results were encountered by other researchers studying ESRD cases and using CRP as a marker for CVD risk. ,,, It is well established that CRP, a marker of inflammation that has been shown in multiple prospective epidemiological studies to predict incident MI, stroke, peripheral arterial disease and sudden cardiac death, in terms of clinical application, seems to be a stronger predictor of cardiovascular events than LDL cholesterol and it adds prognostic information at all levels of calculated Framingham Risk.  cIMT was also higher in ESRD when compared with normal controls. Those results were encountered by many researchers who used cIMT as a surrogate marker of atherosclerosis. , The same results were obtained by Akdag et al in 2008, who found that cIMT, CRP and serum albumin predict long-term mortality in ESRD patients.  Comparison of the lipid profile between both groups in our study resulted in detecting that TGs level is higher and HDL level is lower in group 1 than in group 2. This finding is explained by the fact that the characteristic uremic dyslipidemia consists of normal or low LDL-cholesterol, low HDL-cholesterol and elevated levels of TGs. Correlations were performed within cases and resulted in a positive significant correlation between the relative quantification of TNF-alfa and CRP titer but not the CRP positivity. This can be explained by the fact that in our study the number of patients with ESRD was 51, the percentage of CRP-positive cases was 35.3% and also that among the positive cases for CRP, the range of titer was between six and 96, which is actually in a wide range in addition of denoting high values in some positive cases. The positive correlation between CRP and TNF was detected by other researchers as well.  Another correlation was estimated in our study between cIMT and CRP and resulted in a negative correlation between cIMT and CRP titer among cases but not with those with the CRP positivity. These results can be explained by the wide range of age selected for the study (between 20 and 70 years), the increased upper level of CRP titer mentioned above and the modest upper range of cIMT among ESRD cases (1.3 mm), with a median of 0.7 mm. In a study performed in 2007 by K. Cengiz and D. Dolu, they found that cIMT is positively correlated with age and hsCRP and negatively correlated with serum albumin and serum CRP. 
Other correlations performed in ESRD cases showed that CRP titer showed a strong negative correlation with the age of participants and positive correlation with their HDL levels. The age range in our cases was wide, between 20 and 70 years. Our data highlight the importance of CRP titer and the younger age of the participants in our study. Our results correlating CRP titer to HDL in group 1 cases was positive despite the fact that HDL in our ESRD patients was lower than its level in healthy control subjects. It is known that in patients with ESRD, epidemiological studies have documented that the inverse correlation between HDL-cholesterol and cardiovascular risk is lost and that it does not correlate with the cardiovascular outcome, suggesting probably a dysfunction of HDL. A study by Huang and colleagues in 2010 showed that HDL from ESRD is dysfunctional but can recover to functional HDL after successful renal transplantation. 
We conclude from the results in our study that our patients with ESRD have an increased risk for CVD as denoted by a heightened state of inflammation expressed by increased gene expression of TNF-alfa and CRP titer together with increased atherosclerosis, as expressed by increased cIMT. Both inflammation and atherosclerosis are important denominators of uremic arteriopathy. The importance of TNFalfa gene expression shows the importance of the increased pro-inflammatory cytokine TNFalfa in patients with ESRD, which in turn shows the importance of having to study this gene in our population and its genetic polymorphism as a prognostic TNF-α genetic assay provides a more precise approach for identification of high-risk ESRD patients and can help in the development of accurate and individualized treatment strategies. We recommend that a study similar to this should be carried out in a larger population as a prospective study to evaluate TNF gene polymorphism in ESRD patients on regular hemodialysis before and after renal transplantation.
Conflict of interest: None
We certify that this manuscript nor one with substantially similar content under our authorship has been published or is being considered for publication elsewhere, and all the data collected during the study is presented in this manuscript and no data from the study has been or will be published separately.
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Dr. Mohamed A Marie
Department of Internal Medicine, Faculty of Medicine, Cairo University, Cairo
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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