Abstract
Aim: Oxidative stress (OS) and endothelial dysfunction are implicated in accelerated atherosclerosis in chronic kidney disease (CKD). We assessed endothelial function, OS, and carotid intimal medial thickness (CIMT) and their correlates in 44 CKD stage 5 patients (group III) before and after hemodialysis (HD), 40 patients of CKD stages 3 and 4 (group II), and 25 matched controls (group I). Methods: OS was measured by serum concentration of antioxidants; vitamin C and fractional reducing ability of plasma (FRAP) and pro-oxidant; thiobarbituric acid reactive substances (TBARS). Ultrasonography of carotid artery for CIMT and of brachial artery for flow-mediated dilatation (endothelium-dependent dilatation, EDD) was done. Results: TBARS increased significantly with severity of CKD. Antioxidants FRAP and vitamin C were significantly lower in CKD patients as compared with controls, but there was no significant difference between groups II and III. EDD decreased significantly with severity of CKD, whereas CIMT though higher in CKD patients as compared with controls was not significantly different between groups II and III. After a session of HD as compared with predialysis, levels of TBARS decreased, whereas those of FRAP, vitamin C, and EDD increased. On multivariate analysis, there was negative correlation of TBARS with glomerular filtration rate (GFR), serum albumin, hemoglobin, and EDD. Vitamin C had positive correlations with GFR, serum albumin, hemoglobin, and EDD. EDD had direct correlation with GFR, whereas CIMT correlated negatively with EDD. Conclusions: Endothelial dysfunction and OS occur early in CKD, are closely related to each other and structural atherosclerosis, and are proportional to decline in GFR.
INTRODUCTION
Atherosclerotic cardiovascular disease (CVD) is a leading cause of morbidity in patients on dialysis, with CVD mortality being 30-fold higher in them as compared with general population. Even patients in early stages of chronic kidney disease (CKD) have approximately 2–3 times higher risk of CVD, making CKD a vasculopathic state.Citation1 However, cause of this increase in CVD in patients of CKD is not explained by traditional risk factors alone. Various nontraditional factors such as hyperhomocysteinemia, hyperparathyroidism, inflammation, oxidative stress (OS), and endothelial dysfunction have all been proposed for this increased incidence of CVD.Citation2
Endothelial dysfunction is an early marker of atherosclerosisCitation3 and is known to precede formation of atherosclerotic plaques. Flow-mediated dilatation (FMD), using single resolution ultrasonography, is a reliable noninvasive technique to assess endothelial function, which correlates very well with coronary endothelial function.Citation4
OS due to imbalance between excessive generation of oxidant compounds and decreased antioxidant defense is evidenced by increased levels of compounds formed by interaction of reactive oxygen species with lipids (thiobarbituric acid reactive substances, TBARS), glucose (advance glycation end products), and proteins (advanced oxidation protein products). OS, by causing NO breakdown, can be a major mechanism of endothelial dysfunction.Citation5
Various circulating uremic toxins can be responsible for these abnormalities of endothelial function and OS. If effects of these uremic toxins were rapidly reversible, then their clearance by hemodialysis (HD) would be expected to improve these abnormalities.
Carotid intimal medial thickness (CIMT) represents a reliable marker of structural atherosclerosis. Numerous studies have shown that atherosclerotic disease in carotid system reflects to a high-degree atherosclerotic situation of the coronary artery.Citation6
The present understanding of these important nontraditional cardiovascular risk factors in CKD and HD patients and their interrelationship is limited due to small patient numbers; nonexclusion of patients with comorbidities, which can influence the parameters such as diabetes, smoking, uncontrolled hypertension, and so on; and limited number of studies that have simultaneously assessed all these parameters. With this background, this study was designed to assess endothelial function, OS, and CIMT in various stages of CKD and to examine any relationship between them and glomerular filtration rate (GFR). We also looked at the effect of a single session of HD on these parameters.
MATERIALS AND METHODS
Subjects
Patients with CKD and healthy controls in age group of 18–60 years were included. Group I (n = 25) constituted age- and sex-matched healthy controls who were nonsmokers and nonalcoholic. Subjects with CKD stages 3 and 4 (eGFR 15–45 mL/min/1.73 m2 by MDRD-4 for ≥3 months) were included in Group II (n = 40). Group III (n = 44) constituted subjects with CKD stage 5 on regular HD for ≥3 months. Patients were dialyzed for 4 h with polysulfone hollow-fiber dialyzer using bicarbonate containing HD fluid on volumetrically controlled machines. Urea reduction ratio was calculated in all subjects and was kept at ≥65%. Informed consent was taken from all patients and this study was cleared from the institute's ethics committee.
Exclusion Criteria
Patients having any systemic illness such as diabetes, cardiovascular and peripheral vascular diseases, malignancy, lupus, vasculitis, and so on were excluded. Current smokers and recipients of intravenous iron in the past 4 weeks, patients with hypercholesterolemia (fasting serum cholesterol ≥250 mg/dL), low hemoglobin level (<7 gm/dL), and uncontrolled hypertension (systolic BP > 180 mmHg and diastolic BP > 110 mmHg) were also excluded.
All subjects refrained from caffeine-containing drinks for at least 12 h before start of the study. Angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blocks, statins, and folate were withheld 7 days before the study. Appropriate antihypertensive agents were substituted to optimize blood pressure of the subjects.
Special Investigations
Measurement of oxidants and antioxidants
Ten milliliters of blood collected in plain and EDTA vials was immediately centrifuged at 1250 × g for 10 min and stored at −80°C for subsequent analysis. All samples were light protected to retard deterioration from oxidation of substances during storage. TBARS, markers of lipid peroxidation, were measured by the method of Buege and Aust.Citation7 The method is standardized at our laboratory and a quality control assay was taken to account for the inter- and intra-assay variations. Fractional reducing ability of plasma (FRAP) assay represents total antioxidant capacity of the body. FRAP was assessed by the method described by Benzie and Strain.Citation8 Vitamin C, an important scavenger antioxidant in serum, was measured by the method described by Okamura.Citation9
Measurement of brachial FMD
FMD of brachial artery of limb not having arteriovenous fistula was done using high-resolution B-mode ultrasonography system (P-700, Philips, Eindhoven, The Netherlands) following the procedure described by the American College of Cardiology.Citation5 Group III subjects then underwent a session of maintenance HD after a light nonfatty breakfast. FMD studies were done in these patients within minutes of completion of HD session. Briefly after measuring the baseline diameter of brachial artery,
endothelium-dependent dilatation (EDD) was determined by occluding brachial artery with BP cuff for 5 min and noting maximal diameter 45–60 s after cuff release;
endothelium-independent dilatation (EID) was determined after a rest of 15 min by giving sublingual spray of 400 mcg of glyceral-tri-nitrate and noting the brachial artery diameter after 3 min; and
EDD and EID were expressed as
All measurements were taken at end-diastole coinciding with the R wave on ECG monitor. An average of three consecutive measurements was taken for each subject.
A potential factor that might confound the interpretation of FMD is the baseline diameter. If baseline diameter changes, resulting percentage change in diameter might be affected. Hence, this study also enlisted the absolute change in diameter after intervention (D1 – D0), so as to abolish the above-stated confounding factor.
Measurement of carotid artery intimal thickness
A Philips P-700 ultrasound unit with a 7.5-MHz linear array probe was used for carotid Doppler study. Study was carried out by an experienced radiologist, unaware of patient's history or laboratory findings. Carotid arteries were screened with patient in supine position with neck hyperextended and tilted away from the side to be examined. Evaluation included eight different arterial sites, including common, internal, external, and bifurcation of carotid artery. Mean of intimal media thickness at these eight sites was taken.
Statistical analysis
Statistical analysis was performed on SPSS version 16.0. All data were calculated as mean ± standard deviation. ANOVA was used to compare means between various groups, that is, groups I, II, and III. Post hoc analysis was done with Tukey's test, where applicable. Means of patients of group pre- and postdialysis were compared using the paired t-test. Pearson's correlation coefficient was calculated for various parameters. p-Value of <0.05 was considered significant.
RESULTS
Baseline Characteristics of the Study Population
shows baseline parameters of the three groups, which were age- and sex-matched. There was no difference in diagnosis between groups II and III. Eighty-five percent of patients in group II had a presumptive diagnosis of chronic glomerular disease as compared with 81.8% of patients in group III. There was also no significant difference in BMI and baseline diameter of brachial artery among subjects in all three groups. There was no difference in the mean systolic and diastolic blood pressure between groups II and III.
Table 1. Baseline characteristics of the study population
CIMT and FMD
CIMT (mm) in group I was significantly lower than in groups II and III. We however did not find any significant difference in CIMT between groups II and III.
Results of FMD brachial artery is given in , which shows a statistically significant decline in both parameters of EDD, that is, absolute and percentage change of diameter, across the three groups. There was no difference between parameters of EID in the three groups.
Table 2. Brachial artery flow-mediated dilatation in the study groups
Levels of Oxidants and Antioxidants
depicts the levels of measured oxidants and antioxidants in various groups. A significant rising trend of pro-oxidant TBARS was noted from group I to group III. A progressive declining trend was noticed in level of FRAP from group I to group III, although level of FRAP between groups II and III was not statistically different. Similarly, levels of vitamin C in group I were significantly greater than those of patients in groups II and III, but there was no difference in levels of vitamin C between groups II and III.
Table 3. Levels of various oxidants and antioxidants in the study groups
Effect of Dialysis on FMD and OS Markers
There was a significant improvement in both parameters of EDD after a single session of HD. However, they were still significantly lower than controls and similar to the non-ESRD CKD group ().
Although levels of TBARS showed a significant decline, levels of FRAP and vitamin C showed a significant rise after dialysis (). In spite of this improvement in markers of OS postdialysis, they were still inferior to that of controls. Other than FRAP which is higher in postdialysis group as compared with nondialysis CKD patients (p < 0.001), there was no significant difference between these two groups.
Correlation Analysis for FMD
Of all the parameters studied, on univariate analysis, we found a significant correlation between absolute change in diameter and percentage change in diameter with GFR (r = 0.881 and r = 0.890), vitamin C (r = 0.814 and r = 0.826), FRAP (r = 0.746 and r = 0.764), serum albumin (r = 0.506 and r = 0.518), and hemoglobin (r = 0.746 and r = 0.754). Parameters of EDD however had a negative correlation with CIMT (r = −0.523 and r = −0.535) and TBARS (r = −0.574 and r = −0.576).
On multivariate analysis EDD had a significant direct correlation with GFR (β = 0.451, p < 0.0001) and vitamin C (β = 0.171, p = 0.04) and negative correlation with CIMT (β = −0.163, p = 0.001).
Correlation Analysis for CIMT
On univariate analysis, CIMT had significant negative correlation with GFR (r = −0.419), serum albumin (r = −0.339), hemoglobin (r = −0.358), vitamin C (r = −0.471), and FRAP (r = −0.292) and a significant direct correlation with TBARS (r = 0.230). On multivariate analysis, only a negative correlation with parameters of EDD (β = −0.523, p = 0.001) was significant.
Correlation Analysis for OS Markers
On univariate analysis, TBARS correlated positively with low density lipids (LDL)/high density lipids (HDL) ratio (r = 0.256) and negatively with vitamin C, FRAP, GFR, serum albumin, and hemoglobin (r = −0.586, −0.574, −0.635, −0.360, and −0.462, respectively). FRAP correlated positively with vitamin C, GFR, serum albumin, and hemoglobin (r = 0.807, 0.818, 0.481, and 0.673, respectively) and negatively with LDL cholesterol and LDL/HDL ratio (r = −0.269 and −0.415, respectively). Vitamin C correlated positively with GFR, serum albumin, and hemoglobin (r = 0.848, 0.514, and 0.701, respectively) and negatively with LDL cholesterol and LDL/HDL ratio (r = −0.255 and −0.433, respectively). On multivariate analysis, TBARS had significant negative correlation with GFR (β = −0.546, p = 0.02), FRAP had significant positive correlation with GFR and EDD (β = 0.435, p = 0.01 and β = 0.41, p = 0.04, respectively) and vitamin C had significant positive correlation with GFR (β = 0.435, p = 0.005).
DISCUSSION
We have assessed endothelial function, OS, and the presence of subclinical atherosclerosis in 84 CKD patients (40 CKD stage 3–5 and 44 CKD stage 5D), along with 25 age- and sex-matched controls. We have also looked at the effect of acute HD on these parameters. To establish the effect of CKD per se, we have meticulously excluded common comorbidities such as diabetes, uncontrolled hypertension, CVD, and smoking. Our population was also younger (mean age 31.5 years) and had shorter dialysis vintage (mean duration 9.5 month).
Recently, some studies have shown that impaired endothelium-dependent vasodilatation is a prominent feature in patients with moderate renal impairment,Citation10–12 as well as in patients being treated by HD.Citation13–17 We also document significant blunting of EDD in CKD patients with a graded decline in EDD with the severity of CKD. We found no difference in EID between controls and CKD patients, suggesting that disease process did not significantly affect the vascular wall. Our results are in concordance with previous works of Joannides et al.,Citation16 van Guldener et al.,Citation13 and Ghiadoni et al.,Citation12 who found similarly impaired FMD in 12, 28, and 20 HD patients, respectively. In addition, similar to our observations, Ghiadoni et al.Citation12 found graded decline in FMD with increasing severity of CKD in 40 CKD patients in stages from 3 to 5. Landray et al.Citation11 also found higher levels of plasma von Willebrand factor, a biochemical marker of endothelial dysfunction, in 334 patients with early stages of CKD as compared with controls. They also noted higher levels of von Willebrand factor in patients with increasing severity of CKD. In contrast, although Thambyrajah et al.Citation10 noted significant endothelial dysfunction in patients with mild renal dysfunction, they found similar dysfunction in patients in upper and lower quartiles of GFR. Inclusion of patients having other comorbidities, especially atherosclerotic vascular disease, in their study could be responsible for this discrepancy.
In this study, we also assessed FMD immediately after HD and found a significant improvement in the same. Cross et al.Citation17 assessed the effect of acute HD on FMD in 16 patients at 2, 5, and 24 h post-HD. They found that FMD improved significantly till 5 h after HD. In contrast, Kosch et al.Citation15 in 25 HD patients found an insignificant decline in FMD post-HD. Similarly, Migliacci et al.Citation14 assessed endothelial function in 13 patients having vascular risk factors before and after HD and found no improvement in impaired endothelial function after dialysis. Probable reason behind discordance of these results with ours can be the nonexclusion of diabetic patients and smokers in the study by Kosch et al.Citation15 and inclusion of only high-risk patients by Migliacci et al.Citation14 It is also noteworthy that drugs that have an effect on endothelial function such as ACE inhibitors were not stopped in these studies.
OS, a reflection of imbalance between pro-oxidant and antioxidant mechanisms, is an important cause of increased CVD in CKD patients and has been independently linked to CV events in predialysis CKD patients.Citation18 Although there was no significant difference in FRAP and vitamin C levels between groups II and III taking GFR as a continuous variable, we found an inverse relationship of TBARS and a positive relationship of FRAP with GFR. Similarly, Dounousi et al.Citation19 found that OS appears to increase as CKD progresses and correlates significantly with the level of renal function. OS among HD population has also been reported by other authorsCitation20,Citation21 and has been correlated with inflammatory status and duration of dialysis. However, not many have studied the effect of acute HD on these parameters. We observed a significant decline in levels of TBARS and rise in levels of vitamin C and FRAP after a single session of HD (p < 0.0001), although postdialysis levels were still inferior to that of controls. González Rico et al.Citation21 measured antioxidant enzymes and pro-oxidants in 15 patients before and after HD. Similar to our study, they found significant improvement in these parameters after HD, although even in their study, levels were still poorer than in controls. Himmelfarb et al.Citation22 looked at redox status in 10 HD patients as compared with controls and found that patients had reduced redox status before HD, which got corrected after HD. These findings suggest a dialyzable, rapidly reversible uremic toxin that is responsible for this endothelial dysfunction and pro-oxidant state.
CIMT is a marker of structural atherosclerosis. Increased CIMT is helpful in predicting CV events in population groups.Citation23 Kawagishi et al.Citation24 found increased CIMT in predialysis CKD patients, whereas London et al.Citation25 found CIMT as a contributory factor to mortality in HD patients. In our study CIMT was higher in CKD patients as compared with controls; however, there was no significant difference in CIMT between predialysis and dialysis patients. Dursun et al.Citation26 looked at OS and CIMT in 20 predialysis and 20 HD patients. Similar to our results, they found that patients with CKD had higher CIMT than controls. Same authors reported similar finding in 20 diabetic CKD and 20 diabetic HD patients.Citation27 We found a negative correlation of CIMT with various markers of antioxidant status, that is, vitamin C (r = −0.471), and FRAP (r = −0.292) and a significant direct correlation of CIMT with TBARS (r = 0.230). Similarly, Dursun et al. in both their studiesCitation26,Citation27 also noted significant negative correlation of CIMT with antioxidants and positive correlation with pro-oxidants. We have also looked at correlation of CIMT with FMD of brachial artery, which to the best of our knowledge has not been looked at in CKD and HD population. We found a significant and robust negative correlation between the two on multivariate analysis (β = −0.523, p = 0.001). Recent studies have also shown a significant negative correlation between two patients with coronary artery disease and systemic autoimmune disorders.Citation28,Citation29 The close relationship of CIMT with these nontraditional risk factors and lack of its association with established risk factors such as blood pressure and lipid abnormalities highlight the important role played by these nontraditional risk factors in CKD and HD patients.
There are some limitations of our study. Interpretation of our study is limited by small sample size and cross-sectional design of study. Following the same patients prospectively from predialysis to dialysis stage would determine better the role of increasing renal dysfunction and the effect of dialysis on these parameters. However, our sample size is still larger than most previous studies and we have meticulously excluded comorbidities that frequently occur in patients with CKD and can affect these parameters adversely. We have also studied uniquely the correlation of two important nontraditional risk factors, endothelial dysfunction and OS, with each other and with subclinical atherosclerosis.
CONCLUSIONS
From our results, we can conclude that as compared with controls, patients with CKD(both predialysis and dialysis) have higher OS, greater endothelial dysfunction, and increased structural atherosclerosis. These abnormalities are closely related with each other and with the decline in GFR. Although oxidative and endothelial function improves significantly after HD, they remain lower than that of controls.
Declaration of interest:
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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