1 Chair and Department of Nephrology and 2 Department of Clinical Biochemistry, Jagiellonian University, 15c Kopernika Str., 31501 Cracow, Poland
Correspondence and offprint requests to: Tomasz Stompór, MD, Chair and Department of Nephrology, Jagiellonian University, 15c Kopernika Str., 31501 Cracow, Poland. Email: stompin{at}mp.pl
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Abstract |
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Methods. Of the original cohort of 61 stable patients on PD28 female, 33 male; mean age 50.4±13.6 years; dialyse for a median of 17.5 months at inclusion (range 196 months)47 patients survived the 1 year period on PD. CCA-IMT was assessed at baseline and after 12 months. Pro-inflammatory cytokines (IL-6, TNF), acute phase proteins (CRP, fibrinogen), calcium-phosphate balance and lipid profile were assessed at baseline and after 6 and 12 months. Anthropometric parameters (age, weight, BMI, waist-to-hip ratio) were measured at baseline.
Results. The mean CCA-IMT at baseline, 0.66± 0.19 mm, increased by a mean of 0.098±0.17 to 0.76±0.21 mm (P<0.001) in 1 year. In 14 patients (29.8%) at least one plaque was found in the CCAs examined. At the end of follow-up: 28 patients (59.6%) had increases in CCA-IMT (from 0.63±0.2 to 0.83± 0.21 mm; P = 0.03), and 19 (40.4%) remained stable or even showed slight, but non-significant, decreases of CCA-IMT (from 0.72±0.17 to 0.66±0.17 mm, P = NS). The progressors had significantly higher initial BMI (P<0.05), and mean concentrations of calcium (P = 0.005), IL-6 (P = 0.05), TNF (P = 0.05), CRP (P = 0.005) and lower HDL-cholesterol than non-progressors. In univariate analysis,
CCA-IMT correlated positively with age (R = 0.32, P = 0.03), BMI (R = 0.29, P = 0.05) and mean concentrations of CRP (R = 0.37, P = 0.01), TNF
(0.52, P = 0.0002), but inversely with HDL-cholesterol (R = 0.37, P = 0.01). In multiple regression analysis, however, only age appeared to be independently associated with increase in CCA-IMT (ß = 0.37, P<0.01; R2 for the model 0.14).
Conclusions. Our results suggest a possible role of non-specific inflammation in the progression of atherosclerosis in patients treated with PD, in addition to age.
Keywords: atherosclerosis; chronic inflammation; intima-media thickness; peritoneal dialysis
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Introduction |
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Recently, non-invasive techniques of vascular imaging have emerged which appear to be adequate for assessing atherosclerosis, based on comparisons with findings obtained by invasive techniques or histology [8,9]. These new methodssuch as measuring intima-media thickness (IMT) with ultrasound, quantitatively assessing coronary artery calcium burden with electron beam or multi-slice spiral computed tomographies, or estimating aortic stiffness from aortic pulse wave velocityhave also been proven to predict outcomes for patients in different risk categories.
Among those methods, measuring the common carotid artery (CCA) IMT is the longest applied and the best validated one. The increasing thickness of the intima and media combined, the presence of plaques, and their numbers and sums of thicknesses were shown to be very powerful predictors of adverse outcomes (the onset of cardio-vascular episodes and death) in both the general population and patients with end-stage renal disease (ESRD) [10,11]. CCA-IMT has been shown to be associated with traditional risk factors (such as obesity, lipid profile abnormalities, homocysteine), as well as with those that reflect the state of inflammationfibrinogen, C-reactive protein (CRP), leptin, adhesion molecules, growth factors or serological markers of certain viral infections [10,12,13]. Most of the studies on atherosclerosis and its risk factors performed to date in the setting of ESRD were on patients being treated conservatively (predialysis) or by haemodialysis (HD), or on mixed cohorts [peritoneal dialysis (PD) and HD]. The majority of these studies were cross-sectional in design, although some of them explored the value of baseline IMT for predicting future outcome. Only a few follow-up studies have been done in ESRD patients that focused on trends in this parameter over specific periods of time [10,14]. Recently, an interventional study was published that showed in patients with advanced atherosclerosis a reduction in CCA-IMT after treatment with LDL-apheresis combined with haemodialysis using vitamin E-coated dialysis membranes [15]. To our knowledge no paper has been published to date on changes in CCA-IMT over time in patients treated exclusively with PD.
The aim of the present study was to assess the trends and dynamics of changes in CCA-IMT in a group of stable PD patients over 1 year and to search for possible associations between these changes and the 1 year profiles of selected cytokines, acute-phase proteins and other factors that may be considered risk factors for the development of atherosclerosis, such as anthropometric parameters, calcium-phosphate balance and lipid profile.
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Methods |
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The aetiologies of ESRD in the analysed group were: chronic glomerulopathy in 17 subjects, amyloidosis in four, diabetes mellitus in seven, chronic pyelonephritis and polycystic kidney disease each in four cases, uric acid nephropathy and lithium nephropathy, one case of each. For 23 patients, it was impossible to establish the aetiology of underlying renal disease that led to uraemia, due to late referral to the dialysis unit. The patients were dialysed using Baxter Twin Bag and Fresenius A.N.D.Y Plus or stay safe systems (CAPD) or Baxter HomeChoice or Fresenius PD Night cyclers (APD). Only 21 out of 61 subjects (34.4%) had previously diagnosed ischaemic heart disease based on one or more of typical clinical symptoms or prior ECG or exercise test; and 14 patients (22.9%) were normotensive (blood pressure below 140/90 mmHg and no hypertensive drugs). The dialysis dose of each patient was adjusted to obtain a Kt/V of at least 1.8.
Of the 61 patients, 60 completed the entire study period, one patient died, 47 remained on PD (although five of them were switched from CAPD to APD) and three patients were transferred to HD; 10 patients were transplanted during the study period. We analysed the data on the 47 subjects who were dialysed on PD throughout the study period25 males and 22 females, mean age 52.7±12.8 years, on PD for a median of 16.5 months (range 198 months).
Common carotid artery intima-media thickness
The CCA-IMT was assessed in the B-mode presentation using an Acuson 128 XP/10 with a 5/7 MHz linear transducer (Acuson Corporation, Mountain View, CA). IMT was defined as the distance between the leading edge of the lumen interface and the mediaadventita interface of the far wall, and it was measured on the far walls of the CCAs bilaterally, 24 cm proximal to the bifurcations, and during cardiac diastole. For the purpose of this analysis, the higher of the two results from each side was used. IMT assessment was performed in the plaque-free arterial wall (an atherosclerotic plaque being defined as an echo-structure protruding into the vascular lumen and a thickness greater by at least 50% than neighbouring sites). The method described above has previously been validated by many other investigators [4,10,14,16,17]. All IMT ultrasound studies were performed by the same investigator (A.K.), who was blinded to the patients clinical and laboratory data.
CCA-IMT was assessed at the start of the study (baseline) and after 12 months under the same standardized conditions. In each patient the difference in IMT between these two points of time was calculated (CCA-IMT). Patients in whom the follow-up CCA-IMT was higher than at baseline (positive value of
CCA-IMT) were considered progressors, and the remaining subjects (0 or negative
CCA-IMT) non-progressors.
Anthropometric parameters and blood pressure assessment
The patients were weighed after their abdomen was completely drained of dialysis fluid. Waist and hip circumferences were measured with non-stretchable tape measures, and the waist-to-hip ratio (WHR) was calculated as was the BMI according to standard formula.
Blood pressure was measured at regular outpatient follow-up visits in the sitting position thrice in 3 min intervals, after at least 15 min of rest. The mean value of these three readings was then calculated. A standard sphygmomanometer with a 14 cm cuff was used (Diplomat-Praesameter, Rudolf Riester GmbH, Jungingen, Germany). Pulse pressure (PP) was calculated; and thence the mean arterial blood pressure (MAP) according to the formula: MAP = DBP + 1/3 PP (where DBP is diastolic blood pressure). The details of antihypertensive treatment were also obtained from the patients monthly drug charts.
Laboratory assessment
The fasting blood samples taken for analyses of leptin, cytokines and acute-phase reactants were centrifuged at 4°C at 1000 g for 1530 min. Serum or plasma samples were immediately frozen and maintained at a temperature of 70°C. The serum concentrations of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF), considered pro-inflammatory cytokines, were assessed by the ELISA technique (Quantikine, R&D Systems, Minneapolis, MN). Serum leptin was measured using radioimmunoassay (Human Leptin RIA Kit, Linco Research Inc., St. Charles, MO). The intra-assay variabilities for leptin, IL-6 and TNF
measurements were below 3%.
CRP and fibrinogen were considered positive acute phase reactants. CRP was assayed with the immuno-nephelometric method (N High Sensitivity CRP), and fibrinogen using Multifibren U reagent (Dade Boehring Marburg GmbH, Marburg, Germany). The Behring II Nephelometer was used for both measurements (Dade Behring Marburg GmbH). Serum albumin was considered a negative acute-phase reactant and nutritional marker; it was measured with a Hitachi 917 analyser (Roche Diagnostics, Division of Hoffmann-La Roche, Basel, Switzerland; this analyser utilizes the bromcresol green method for albumin assay). All of the mentioned analyses were performed in duplicates.
All patients had measurements of total serum cholesterol (total cholesterol), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides (TG), calcium and phosphate serum levels using a Hitachi 917 analyser. Serum intact parathyroid hormone (iPTH) was assayed in EDTA-anticoagulated plasma with chemiluminescence using the Immulite 2000 kit (Diagnostic Products Corporation, Los Angeles, CA). Haemoglobin and haematocrit were assayed with Sysmex XE-2100 equipment (Roche Diagnostics).
The dialysis and residual components of Kt/V and wClCr both were calculated based on 24 h urine and dialysate collections performed prior to the scheduled visit to the PD unit. Serum, urine and dialysate urea and creatinine levels were analysed using the Hitachi 917 equipment. Calculations of Kt/V as well as wClCr were performed using original Polish software, Nephron 97 for Windows (DDPS, Cracow, Poland). With this software, urea distribution volume is calculated according to the Watson formulas. Residual renal function was expressed as the mean value of the sum of the residual urea and creatinine clearances. The peritoneal equilibration test was performed according to the method described by Twardowski.
All biochemical and inflammatory parameters (the term inflammatory as used in this paper also being applied to leptin) were measured at the beginning of the study and 6 and 12 months after that. If the patient had any inflammatory, infectious or CVS episodes, blood sampling was delayed by 1 month after its complete resolution. At the end of the study the mean concentrations of the analysed parameters from the three measurements were calculated in an attempt to reflect the patient's 1 year profile of exposure to risk factors. Changes in IMT were analysed when applicable in relation to the mean values of tested parameters and absolute changes in their concentrations between baseline and end of follow-up.
The study protocol was reviewed and accepted by the independent ethical committee of the Jagiellonian University.
Statistical analysis
Statistical analysis of the data was performed using the Statistica 5.1 software (StatSoft Inc., Tulsa, OK). Shapiro-Wilk's W test of normality was used to analyse data distribution. All variables that were distributed normally are presented as mean±SD, those with non-normal distribution, as median and range. For correlations between variables, the Pearson test was used for normally distributed data and the Spearman R test for those with non-parametric distributions. For inter-group comparisons, we used the Student t-test for normally distributed variables or the MannWhitney U test for non-normally distributed data. Depending on data distribution, the comparison of follow-up data vs baseline was performed using the Student t-test for dependent variables or the Wilcoxon test. To assess the influence of tested parameters on CCA-IMT as the dependent variable, backward linear multiple regression analysis was performed. We considered P values below 0.05 as statistically significant.
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Results |
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Discussion |
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As in our paper, most of the studies report a highly significant association between the prevalence of plaques detected by ultrasound and CCA-IMT, and when applicable, a close association between the sum of thicknesses of plaques (plaque score) and IMT [4,10,14,17,20].
In our study, age was significantly correlated with CCA-IMT, and it was the only parameter that multiple regression analysis identified as being independently associated with IMT. Diabetics were also more prevalent in the progressors group. Almost all other papers on the topic confirm the significance of age and diabetes in predicting CCA-IMT in ESRD patients [4,17,1922]. In our study a marginally significant difference in dialysis vintage between progressors and non-progressors was found. Most of the studies in this area have not confirmed the impact of dialysis vintage on CCA-IMT value [4,17,23] (although there are also some papers available that have documented such an association [24,25]).
The groups with and without progression in CCA-IMT differed in mean serum calcium level; the Ca x P product was borderline higher in the progressors, and there was no differences were shown in phosphate levels. Similar results were obtained recently in another study of patients treated exclusively with PD. Okhuma et al. [18] found statistically significant association between CCA-IMT and serum calcium level, whereas no such association could be demonstrated for phosphates, Ca x P product or iPTH. Other studies of ESRD patients did not, however, demonstrate any association between IMT and any parameter of calcium-phosphate balance [19,22]. In contrast, Oh et al., who analysed factors that may affect IMT in young adults with childhood-onset renal failure, found associations between CCA-IMT and the cumulative concentrations (i.e. the mean of all results available since the onset of disease) of calcium, phosphate and Ca x P product [21].
As it is well recognized, the lipid profile considered to be atherogenic in the general population still is atherogenic in patients on maintenance dialysis; however, its impact on survival appears to be paradoxical and exemplifies the phenomenon defined recently as reverse epidemiology [3]. Those patients from our cohort whose IMT progressed were characterized by significantly lower mean serum HDL-cholesterol and a significant decrease in HDL-cholesterol within the study period; serum HDL-cholesterol was also inversely associated with CCA-IMT in univariate analysis. This relationship existed despite the fact that 70.2% of the studied patients were treated with lipid-lowering drugs, mostly statins. In a cross-sectional study of patients treated exclusively with PD, significant correlations were found between total cholesterol, LDL-cholesterol and CCA-IMT, but these associations were not confirmed further in multivariate analysis [18]. Drüeke and co-workers [22] demonstrated an independent association between LDL-cholestrol and CCA-IMT in 71 patients aged 56±16 years who were on HD for 76±75.5 months along with a similar association between CCA-IMT and triglycerides in the subset of patients over 60 years old. However, several studies report no significant associations between lipid profile and CCA-IMT in ESRD subjects, although some of them suggest such an association with lipoprotein (a) [4,18,20,21].
Several significant correlations emerged between CCA-IMT and mean CRP and TNF
levels, calculated from three measurements, and
TNF
over 1 year; similarly, progressors and non-progressors differed significantly in mean serum levels of CRP, IL-6 and TNF
over the entire observation period, as well as in
TNF
over the same interval. The relationship of IMT with TNF
and
TNF
deserves special attention since this cytokine is considered a key substance in the inflammation-mediated vessel wall damage of atherosclerosis. Its atherogenic properties include the activation of macrophages that infiltrate vessel wall, the enhancement of the local expression of metalloproteinases and adhesion molecules, and the stimulation of CRP synthesis. The expression of scavenger receptors is also controlled by this cytokine. In addition, acting as a potent inhibitor of lipoprotein lipase, TNF
mediates the generation of the substrate for foam cell formation [26].
An association between CCA-IMT and markers of inflammation was addressed previously by other authors. Okhuma and co-workers [18] found statistically significant correlations between IMT, plaque score, and CRP, fibrinogen, TNF soluble receptor and IL-1 receptor antagonist. Patients within successive CRP tertiles differed significantly in both IMT and plaque score. In the multiple regression model, both investigated features of CCA were independently associated with CRP. Among the patients with advanced renal failure not yet on dialysis, whom Stenvinkel et al. [27] studied, those displaying symptoms of malnutrition were characterized by significantly higher values of the three different parameters derived from CCA ultrasound ([IMT, CCA diameter and the calculated intima-media area (cIMA) and higher serum CRP and fibrinogen]. The mean values of CCA and cIMA were also significantly higher in subjects with serum CRP above 10 mg/l than in those with CRP below 10 mg/l. The patients with at least one plaque in their CCA were also characterized with higher CRP and TNF and lower serum albumin than those who were plaque-free [27]. Albumin was also shown to be inversely and independently associated with CCA-IMT in some studies of patients treated with PD or HD [17,23]. Although there may be many different clinical circumstances that lead to hypoalbuminaemia in ESRD, one convincing explanation for the mentioned association is that albumin may represent the state of enhanced inflammatory response as a negative acute-phase protein. We, however, found no difference in serum albumin between progressors and non-progressors; we also did not find any association between
CCA-IMT and mean serum albumin or the change in its serum level, despite the fact that albumin concentration decreased significantly during the 1 year follow-up. The significant positive correlations between IMT, the sum of thicknesses of CCA plaques and concentrations of circulating adhesion molecules that studies of patients treated with both HD and PD found additionally support an inflammatory concept of atherogenesis [4,23].
As we mentioned in the introduction to this paper, only limited data exist on changes in CCA over time detected by ultrasound of ESRD patients. In one of follow-up studies, Stenvinkel et al. [27] analysed this topic in patients with advanced renal failure shortly before the initiation of renal replacement therapy. In a group of 45 patients, they identified two subgroups: 22 patients in whom the calculated cross-sectional intima-media area (cIM) progressed over one year and 23 who remained stablealthough no progression was noticed for the group as a whole. The patients who progressed did not differ from the non-progressors in terms of baseline age, BMI, prevalence of cardio-vascular disease, diabetes, malnutrition, serum albumin or lipid profile. The patients who progressed had, however, a significantly higher baseline IL-6 concentration (P<0.05) and prevalence of IgA antibodies against Chlamydia pneumoniae (59 vs 17%; P<0.01). Similarly, patients with at least one CCA plaque had higher serum IL-6 than plaque-free subjects (6.0 vs 2.5 pg/ml, respectively; P<0.001). The increment observed over a 1 year follow-up in cIM was associated with the initial IL-6 (r = 0.41, P<0.01); log-transformed IL-6 concentration was the only variable that independently predicted this increment. Patients with high initial serum IL-6 (>10 pg/ml) were characterized by a significantly higher increase of cIM [14].
In another study that repeatedly assessed IMT in 90 subjects on long-term HD, the baseline CCA-IMT was associated with age, systolic blood pressure, serum CRP and markers of endothelial dysfunction, serum homocysteine and asymmetric dimethyl-arginine (ADMA). In this cohort, the increment in CCA-IMT observed after 15.1±1.1 months was associated with the initial serum CRP and ADMA concentrations. Interestingly, the initial IMT was significantly and inversely correlated with CCA-IMT over the follow-up period (r = 0.41; P = 0.001) [10]. Our results are very similar: in our patients the initial IMT is also inversely correlated with
IMT (R = 0.31; P = 0.03), and its initial value was lower in the progressors than in the non-progressors, although not significantly (0.63±0.2 vs 0.72±0.17, respectively). This may suggest that patients who initially have advanced atherosclerosis tend to relatively stabilize over time, and those with lower initial values tend to progress.
We should emphasize the very significant absolute increase in IMT after 1 year in our entire cohort (by 0.098±0.17 mm), and particularly in the patients who progressed (0.2±0.14 mm). Most of the studies in this area report an annual increase ranging between 0.04 and 0.05 mm. To our best knowledge, no follow-up study has been published or performed to date on patients treated with PD only, therefore, we can make no direct comparisons with other cohorts. Such a high progression in IMT probably reflects the highly expressed risk profile in our cohort of patients, but the atherogenic nature of PD as renal replacement therapy cannot be ruled out.
We are aware of several limitations in our study, such as its relatively small sample size, short-term follow-up and its observational design. Nevertheless, in our opinion our results may add important information to the knowledge concerning progression in carotid atherosclerosis in PD patients.
In summary, we would like to emphasize that our study appears to be the first one in the available literature to provide data on the repeated measurement of CCA-IMT exclusively in patients on PD and that correlates these with traditional and inflammatory risk factors for atherosclerosis collected not only at baseline, but also repeatedly over a follow-up period. Unlike in several, cross-sectional studies performed previously in different ESRD cohorts, our results did not confirm any independent association between the progression of atherosclerosis and chronic inflammation, or with other traditional risk factors, except for age. One may doubt that age is a factor sufficient as a single variable to underlie such a significant progression. However, there are still many other variables that might have an impact on the progression of atherosclerosis, which we did not analyse in our present study. It appears to be noteworthy that, even in a group of individuals with high-risk profiles for the progression of atherosclerosis, such as ESRD patients on PD, there are individuals who display no increase in CCA-IMT.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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