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*
Servicios de Nefrología, Hospital Valle
Hebrón, Barcelona, Spain.
Bioquímica, Hospital Valle
Hebrón, Barcelona, Spain.
CDR Monolab, Hospital Sant Gervasi, Barcelona, Spain.
Unidad de Hemodiálisis, Hospital Sant
Gervasi, Barcelona, Spain.
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Centro de Nefrología, Virgen de
Montserrat, Barcelona, Spain.
¶
Centro de Diálisis Nephros, Barcelona,
Spain.
Correspondence to Dr. Alfons Segarra Medrano, Unidad de Investigación, Servicio de Nefrología, Hospital Valle Hebrón, Passeig Vall d'Hebrón, 119-129, E-08035 Barcelona, Spain. Phone: 34-93-274 61 52; Fax: 34-93-274 62 04; E-mail: asem{at}hg.vhebron.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, lipids,
apoproteins A1 and B, and albumin were measured in a group of 200 nondiabetic
dialysis patients and 100 healthy matched volunteers. When compared with
healthy controls, dialysis patients showed increased levels of CRP, TM, TPA,
and PAI-1 and evidence of increased thrombin-dependent fibrin formation.
Increased levels of active PAI-1 were associated to a great extent with major
classic vascular risk factors and to a lesser extent with CRP and serum
triglycerides. Forty-six patients (23%) had evidence of CAD. Variables
associated with CAD in the univariate analysis included age, time on dialysis,
male gender, number of packs of cigarettes per year, high BP, fibrinogen,
apolipoprotein B, albumin, PAI-1 activity, CRP, thrombin-antithrombin
complexes, and fibrinopeptide A. Logistic regression analysis found age,
high-density lipoprotein cholesterol, gender, high BP, CRP, time on dialysis,
and PAI-1 activity to be independent predictors of CAD. This model classified
correctly 85% of patients as having CAD and showed adequate goodness of fit
for all risk categories. Our data support a pathogenic link among activated
inflammatory response, endothelial injury, and CAD in hemodialysis patients
and suggest that assessment of circulating PAI-1 levels could be an additional
tool to identify dialysis patients who are at risk for developing atheromatous
cardiovascular disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was designed (1) to investigate the relationship between the circulating levels of the endothelial cell glycoproteins PAI 1, TPA, and TM and the major vascular risk factors described in dialysis patients and (2) to determine the role of these endothelial cell products as independent predictors of CAD in a large group of nondiabetic dialysis patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cause of chronic renal failure was glomerular disease in 70 patients (35%), interstitial nephritis in 30 patients (15%), polycystic kidney disease in 35 patients (17.5%), vascular disease in 20 patients (10%), and unknown in 45 patients (22.5%).
The control group comprised 100 healthy, age- and gender-matched volunteers who were recruited from among the population of healthy people living in the geographic area of our center.
The study protocol was accepted by the ethics committee of our hospital, and all patients gave their written informed consent before participation.
Laboratory Procedures
For lipid and apoprotein assays, blood was collected from the antecubital
vein in glass tubes with no additives after an overnight fast of 12 h before
the start of a dialysis session. For the hemostasis and fibrinolysis assays, a
19-gauge needle with no tourniquet was used to collect 10 ml of blood into
silicone tubes that contained 0.3 ml of 3.8% sodium citrate. Samples were
cooled in ice water and centrifuged immediately at 1000 x g for
45 min at 4°C. The platelet-poor plasma so obtained was aliquoted and
stored at -80°C until assayed within a 1-mo period.
High-density lipoprotein (HDL), low-density lipoprotein, and very-low-density lipoprotein were isolated by sequential ultracentrifugation. In the three lipoprotein subfractions, cholesterol and triglycerides were determined by enzymatic methods (cholesterol, CHOD-PAP; triglycerides, glycerol-3-phosphate-oxidase-peroxidase; Boehringer Mannheim, Mannheim, Germany). Apoprotein A1 (apo A1) and apo B were determined by nephelometry (Beckman Array System; Beckman, Fullerton, CA; interassay CV 7% for apo A1 and 4.5% for apo B).
Serum levels of PAI-1 Ag, TPA Ag, D-dimer, thrombin-antithrombin (TAT)
complexes, TPA/PAI complexes, and TM were determined by enzyme-linked
immunosorbent assay (ELISA; Asserachrom Diagnostica Boehringer Mannheim,
Mannheim, Germany). TPA and PAI-1 activities were determined by chromogenic
assays (Biopool, Umea, Sweden). Fibrinopeptide A (FPA) was determined by ELISA
(Boehringer Mannheim GmbH; Diagnostica Stago). C-reactive protein (CRP) was
determined by the Behring Nephelometry immuno-assay (NA latex CRO, Behring
Institute, Galway, Ireland). Serum interleukin-1ß (IL-1ß) and tumor
necrosis factor- (TNF) were determined by ELISA (Medgenix
Diagnostics, Brussels, Belgium). Albumin concentration was determined by the
green bromcresol method.
All assays were performed in duplicate and calibrated with purified standards and reference plasmas from the manufacturers. Intra- and interassay coefficients of variation for all tests were determined with the use of 20 different plasma samples.
To avoid the influence of acute intercurrent diseases on the biochemical parameters, we carried out analyses at least 6 mo after the clinical event in all patients who required hospitalization for acute illness or infections (n = 6) or who had surgical procedures (n = 4), episodes of kidney allograft rejection (n = 2), or acute vascular thrombosis (n = 1) before being entered inthe study.
A prerequisite for including the levels of endothelial cell glycoproteins,
CRP, and inflammatory cytokines in statistical analyses was to demonstrate
that the predialysis levels of all of these variables showed little variation
over time when measured in the same group of patients. For this purpose, we
analyzed the variation coefficients for the different variables in 30 random
patients before the start of four consecutive dialysis sessions. They were as
follows: TPA Ag, 12%; PAI-1 Ag, 16%; PAI-1 activity, 14%; FPA, 8.4%; TAT
complexes, 12.3%; CRP, 11%; IL1-ß, 26%; TNF-, 20%; and soluble TM,
9.8%.
Clinical Data Collection
Information on risk factors was obtained by medical record review, personal
interview, and physical examination. Coronary angiography was performed on all
patients who had no previous episodes of well-documented acute myocardial
infarction and who experienced clinical symptoms that suggested ischemic heart
disease associated with ST changes in the ECG.
Definitions
CAD was diagnosed in the presence of one of the following: (1)
definitive episode of myocardial infarction with appropriate rise in serum
creatinine phosphokinase associated with typical ECG signs of necrosis (Q
waves) or confirmed with technetium pyrophosphate nuclear cardiac scan,
(2) previous coronary bypass surgery or coronary angioplasty, or
(3) evidence of >50% coronary artery stenosis as determined by
selective coronary angiography.
Smoking was assessed by personal interview and entered in the analysis in two ways: (1) as current smokers versus others and (2) as a quantitative variable: packs per year (number of packs per day multiplied by years of smoking).
Patients were considered hypertensive when they were receiving antihypertensive treatment or when three resting predialysis measurements showed diastolic BP >90 mmHg or systolic BP >140 mmHg.
Body mass index was calculated as weight divided by height, squared.
Statistical Analyses
Results are given as the mean ± SD. Differences in clinical and
biochemical risk factors between groups were calculated by unpaired t
test. Qualitative variables were compared with the use of the
2 test. Correlation analyses among quantitative variables were
carried out with Pearson's correlation test. A P value of less than
0.05 was considered statistically significant. To analyze the factors that
determined the plasma concentration of endothelial glycoproteins, we carried
out single regression analyses introducing the dependent variables (FPA, PAI,
CRP, TM, and D-dimer) after logarithmic transformation. All of the variables
with P values less than 0.1 in single regression analysis were
entered into a stepwise multilinear regression analysis. We determined the
most parsimonious model by removing single variables.
To determine which variables were associated independently with CAD, we carried out a univariate analysis that compared patients with and without CAD. All of the variables with P values less than 0.1 in the univariate analysis were entered into stepwise multiple logistic regression analysis with a forward selection method. PAI activity, CRP, and fibrinogen were tested in the model both as continuous variables and after being categorized into four groups, each determined by the cutoff quartile points. Odds ratios (OR) were calculated from the regression coefficients as an approximation of the relative risk. The linearity of OR was analyzed to determine whether there was a continuous relation between the risk factors and clinical event. To examine a possible effect modification, we tested the interaction terms of PAI-1 activity with fibrinogen and CRP.
Once obtained, the predictive logistic model was tested blindly on an independent group of 60 dialysis patients. This group was obtained randomly from among 150 patients who had been dialyzed in two outpatient dialysis centers that did not participate in the first phase of the study and consisted of 40 men and 20 women, aged 40 to 75 yr (62.5 ± 24 yr), dialyzed three times a week for 10 to 125 mo. The hemodialysis prescription was 9 to 13.5 h/wk with the use of a 1.2- to 1.6-m cuprophane hollow-fiber filter and bicarbonate dialysate containing 2 g/L glucose. Forty-two patients (70%) received erythropoietin therapy. Information on risk factors was obtained by medical record review, and blood samples were obtained before a hemodialysis session. An independent group of researchers who were blinded to biochemical results defined the presence of CAD according to the criteria defined previously.
Hosmer-Lemeshow's test (16) was used to calculate the discrimination power and goodness of fit of the logistic model. Statistical analyses were performed with the Statistical Package for the Social Sciences for Windows, 6.1.2 (SPSS, Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and soluble TM. In contrast, serum levels of HDL
cholesterol, apo A1, and albumin were significantly lower
(Table 1). When the patients
were classified according to the disease that led to end-stage renal failure,
patients with renal disease of both vascular and unknown origin were older
than the remaining groups (P < 0.01). There were, however, no
significant differences among groups for any of the biochemical variables
analyzed.
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Table 2 summarizes the matrix of Pearson correlations among the endothelial cell-derived molecules, fibrinolysis, and hemostasis parameters. Table 3 summarizes the results of stepwise multiple regression analyses carried out to determine the independent predictors of circulating levels of endothelial glycoproteins and certain hemostasis parameters.
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We found a close correlation among CRP, IL-1ß, and TNF
(P < 0.01), indicating that these three variables probably were
measuring the same process. In addition, when determined prospectively, CRP
showed lower variability than IL-1ß and TNF. Therefore, we
selected CRP as the most representative variable that indicated an activated
acute-phase response. CRP correlated significantly with age (r, 0.23;
P < 0.01), fibrinogen (r, 0.52; P < 0.01), and certain
fibrinolysis variables (see Table
2) and correlated negatively with both HDL cholesterol (r, -0.19;
P < 0.05) and albumin levels (r, -0.44; P < 0.01).
When the logarithm of CRP concentration was considered as a dependent variable
in a forward stepwise multiple regression analysis, albumin, age, fibrinogen,
and PAI-1 activity were the only variables accepted in the final equation and
accounted for 18% of the variability of CRP levels (r2, 0.18;
P < 0.001).
Forty-six patients (23%) had evidence of CAD as diagnosed by previous coronary bypass or coronary angioplasty (n = 11), definitive myocardial infarction (n = 18), or coronary angiography (n = 17). Table 4 shows the clinical and biochemical characteristics of the patients with and without CAD. The clinical variables associated with CAD in the univariate analysis were age, time on dialysis, male gender, number of packs of cigarettes per year, and high BP. Fibrinogen, apo B, PAI-1 Ag, PAI-1 activity, CRP, TAT, and FPA all were significantly higher in the patients with CAD, whereas serum HDL cholesterol, apo A1, and albumin all were significantly lower. Soluble TM was not associated with CAD in the univariate analysis.
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The final logistic regression model obtained after a forward selection of
the variables included age, HDL cholesterol, gender, high BP, CRP, PAI-1
activity, and time on dialysis as independent predictors of patient status
(Table 5). This model correctly
classified 85% of patients as having CAD and showed an adequate goodness of
fit for all risk categories (2, 1.21).
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Although the degree of association between CRP and CAD in the univariate
analysis was very similar to that observed between albumin and CAD, in the
multivariate analysis albumin concentration lost its predictor significance
when CRP was introduced in the model. Moreover, among the variables determined
to analyze activation of the inflammatory response, CRP showed the highest
statistical significance as predictor of coronary stenosis. When IL-1 or
TNF were introduced instead of CRP, the predictive model, although
significant, lost discriminative capacity and showed an inadequate goodness of
fit. That was particularly evident for the low-risk categories (area under the
curve, 0.77 [P < 0.01]; 2, 10.32 [P <
0.01]). The goodness of fit of the model was significantly better when values
for CRP, PAI-1, and FPA were introduced as continuous quantitative variables
rather than as categorical variables defined by their corresponding quartiles
(2, 1.21 versus 6.12, 7.1, and 9.45, respectively;
P < 0.001). Finally, after having obtained the main effects model,
we tested the interaction terms of PAI-1 activity with fibrinogen, CRP, and
FPA, which were consistently nonsignificant.
The prevalence of CAD in the independent group of patients selected to validate the model was 28.3% (n = 17). There were no significant differences in any of the clinical or biochemical parameters between this group of patients and the 200 patients studied in the previous phase. The logistic model classified correctly 82.35% of patients in this group for having CAD, and goodness of fit also was adequate for all of the risk categories (Table 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with previous reports (9,10,11,12,13), our dialysis patients showed increased circulating levels of endothelial glycoproteins. Although we cannot exclude the possibility that individual patients who exhibited increased levels of these endothelial products might have experienced subclinical infections or imperceptible vascular complications, our method was designed to guarantee that the parameters studied were not influenced by acute intercurrent events in the vast majority of patients. Moreover, our patients showed features consistent with an activated acute-phase response. The plasma levels of CRP, IL-1, and TNF tended to remain constant over time, suggesting that the activation of acute-phase response was not due to acute intercurrent diseases.
The first relevant finding of our study was the evidence of a significant statistical association between levels of both PAI-1 Ag and TPA Ag and major vascular risk factors. This association supports the hypothesis that the increase in circulating levels of these molecules was due, at least in part, to direct injury of endothelial cells. Moreover, to a lesser extent, levels of both PAI-1 Ag and TPA Ag—but not TM—correlated significantly with CRP and other inflammatory markers. This association has been described in other studies and indicates that PAI-1 is an acute-phase protein that can rise in response to several stimuli, including cytokines such as IL-1 and TNF. An alternative explanation for the relationship between PAI-1 and CRP is that the increase in circulating PAI-1 reflected a cytokine-mediated or -facilitated endothelial cell injury as supported by certain clinical and experimental data (17,18,19,20). The association between PAI-1 and serum triglycerides observed in our group of patients is in agreement with data reported previously in both dialysis and nondialysis patients (12,15,21). This association is supported by recent experimental data indicating that very-low-density lipoproteins induce PAI-1 synthesis through a signaling pathway involving protein kinase C-mediated mitogen-activated protein kinase activation (22). This pathway could be of relevance in dialysis patients because the increase in very-low-density triglycerides is a common feature of uremic dyslipidemia (23).
Although the increase in soluble-free TM has been considered classically as a biochemical marker of endothelial injury, our findings are not in concordance with this hypothesis because TM was associated neither with major vascular risk factors nor with coronary heart disease. This lack of association probably indicates that TM is not a reliable marker of endothelial cell injury in chronic renal failure because a significant amount of the increase in circulating TM is due to the loss of renal function itself (24,25).
The second interesting point of our study was the evidence that most of PAI-1 was circulating in its active form with the potential ability to bind and inactivate TPA. This increased PAI-1 activity might explain the discrepancy that we observed between TPA concentration and TPA activity. To analyze the functional significance of increased PAI-1 activity on intravascular fibrin breakdown, we measured plasma levels of FPA, TAT complexes, and D-dimer. In agreement with other studies (26,27,28), our patients showed higher levels of TAT complexes and FPA than healthy control subjects. FPA, a biochemical marker of intravascular fibrin formation (29), was associated significantly with fibrinogen and TAT complexes, indicating an increased intravascular thrombin-dependent fibrin formation. Moreover, D-dimer was associated negatively with PAI-1 activity, which is concordant with a certain degree of hypofibrinolysis that was evident even in the absence of clinical manifestations of intravascular thrombosis.
To determine the role of endothelial cell products as independent predictors of CAD, it was necessary to establish objective criteria to define CAD. In dialysis patients, the prevalence of ischemic heart disease resulting from coronary artery stenosis is difficult to determine because clinical angina with no CAD occurs in 30 to 50% of cases and a number of patients may have silent coronary stenoses (30,31). To avoid false-positive results, we established a restrictive operational definition of ischemic heart disease, requiring unequivocal evidence of coronary stenosis. According to these definition criteria, the prevalence of CAD observed in our patients was similar to that described in previous studies (30,31,32,33,34,35).
The third and more relevant finding of our study was that an increased circulating level of active PAI-1 was an independent predictor of coronary artery stenosis in nondiabetic dialysis patients after adjusting for CRP and other major vascular risk factors. It should be emphasized that the multivariate logistic model obtained had a high predictor capacity and goodness of fit, both in the large series studied and in the validation series, indicating that it could be of potential applicability in clinical practice for estimating the risk of CAD.
The independent association between CRP levels and vascular disease
observed in our patients concurs with data from several authors
(36,37,38,39,40,41,42).
Of all of the inflammatory markers analyzed, CRP showed the highest
statistical significance as a predictor of coronary artery stenosis. When
albumin, IL-1, or TNF were introduced instead of CRP, the model,
although significant, lost discriminative capacity and showed an inadequate
goodness of fit that was particularly evident for the low-risk categories.
The independent association between circulating PAI-1 and the presence of coronary artery stenosis had not been described previously in dialysis patients. In the general population, elevated PAI-1 has been shown to be unvariably predictive of unstable angina and myocardial infarction only in patients with preexisting CAD (43,44). In comparison with these observations, population-based studies have demonstrated an inconsistent association between fibrinolytic parameters and the development of CAD. The Caerphilly Study (45) found that PAI-1 activity was not predictive of incident CAD, whereas the Prime Study (46), a prospective cohort study of 10,500 men who initially were free of cardiovascular disease, noted a significant association between the PAI-1 level and cardiovascular disease after adjusting for other cardiovascular risk factors. Theoretically, PAI-1 could be related with atheromatous disease either as a passive marker of an endothelial cell injury of multifactorial origin or as a potential inductor of an hypofibrinolysis state, which may permit fibrin to persist on the surface of the injured vessel. Our data account only for a small percentage of circulating PAI-1 variability but support the hypothesis that in dialysis patients, circulating levels of PAI-1 increase in part as a result of endothelial cell injury and may contribute to the atheromatous disease promoting a certain degree of hypofibrinolysis.
In summary, in this study we provided evidence that, in dialysis patients, the circulating levels of the endothelial cell glycoproteins PAI-1 and TPA were statistically associated with major vascular risk factors and, to a lesser degree, with an activated acute-phase response and serum triglycerides. Moreover, circulating PAI-1 was an independent predictor of coronary artery stenosis after adjusting for the major vascular risk factors and CRP. Taken together, our data suggest that increased circulating PAI-1 could indicate a chronic endothelium activated state and could be an additional tool to identify dialysis patients who are at risk for developing atheromatous cardiovascular disease. Future studies designed to define further the underlying pathways that account for increased circulating PAI-1 levels would be of great interest to delineate the pathogenic mechanisms that lead to ischemic cardiovascular disease in this group of patients.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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