Department of Nephrology and Internal Medicine, Medical Academy, Bialystok, Poland
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Abstract |
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Methods and results. sTM levels of 10.7 (5.7230.7) ng/ml in 100 HD patients were higher than in 30 controls (P<0.0001). In a bivariate regression analysis, immunoreactive sTM was positively associated with the presence of hepatitis B virus surface antigen and/or anti-hepatitis C virus antibodies measured by third generation ELISAs (P<0.0001), and was related to certain markers of liver injury and biosynthetic dysfunction. sTM was also directly associated with time on dialysis (P=0.001), or use of unfractionated heparin (UFH) (vs enoxaparin) (P=0.0007), erythropoietin (P=0.008), ACE-inhibitors (P=0.034), acetate-buffered dialysate (vs bicarbonate) (P=0.040), pre-dialysis systolic (P=0.012), and diastolic blood pressure (P=0.043). It was negatively associated with lipoprotein(a) (P=0.029). sTM was not related to age, sex, smoking, cause of renal failure, prevalence of cardiovascular disease, amount of HD delivered, preserved residual renal function, ferritin, C-reactive protein, and other vasoactive medications used. In a multivariable analysis, a positive hepatitis marker (P=0.0002), the use of UFH (P=0.030) and erythropoietin (P=0.019), and raised pre-dialysis blood pressure (P=0.024) were positive independent predictors of high sTM level.
Conclusion. These data indicate that, in addition to endothelial activation, elevated sTM levels in HD patients may be related to viral infection and/or liver dysfunction, and influenced by modifiable factors such as increased blood pressure, and the type of heparin and erythropoietin treatment used.
Keywords: arterial blood pressure; erythropoietin; haemodialysis; heparin; soluble thrombomodulin; viral hepatitis
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Introduction |
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During vascular injury, the endothelium-bound TM can be cleaved by various proteases and its extracellular portion is released into the blood and/or undergoes internalization and degradation by EC [1]. These events along with tissue factor expression turn the previously thromboresistant EC layer into a procoagulant surface. Soluble TM (sTM), which retains anticoagulant properties [1], is cleared from circulation by the kidneys, and is probably degraded by the liver. The physiological role of sTM is unknown, but has been traditionally regarded as a marker of vascular EC injury in a variety of disorders [2,3]. Recently, the Atherosclerosis Risk in Communities Study group revealed decreased risk of coronary heart disease and less prevalent asymptomatic carotid artery atherosclerosis in patients with high sTM levels [4]. Another prospective study showed a higher incidence of haemorrhage in patients with increased sTM levels receiving oral anticoagulants [5]. It has been suggested that these phenomena are the result of increased synthesis and membrane expression of TM followed by its release rather than due to EC damage [4]. This could call into question previously developed paradigms that elevated levels of sTM point exclusively to a prothrombotic and atherogenic state.
EC activation and injury have been demonstrated in maintenance haemodialysis (HD) patients, and have been causally linked to haemostatic defects, accelerated atherogenesis, and increased cardiovascular mortality [68]. Among the various markers of this condition, elevated sTM levels have been repeatedly found in HD patients. Although this elevation is partially a result of the lack of renal excretory function [810], increased sTM levels have also been reported to be a marker of EC injury in HD patients. sTM has been correlated with prolonged skin bleeding time and defective platelet aggregation [8], increased levels of another EC marker, plasminogen activator inhibitor-1, and reduced ability of EC to release their components upon activation [8], as well as with a longer duration of HD therapy [6,7,11]. Elevated sTM has also been found to be predictive of previous blood access thrombosis in chronic HD patients [12].
The aim of this study was to determine factors that may be predictive of sTM levels in long-term HD patients. Of the factors that have not been examined in this population, we assessed the relationships between sTM levels and viral hepatitis, certain HD treatment-related variables, residual renal function (RRF), the use of recombinant erythropoietin (rHuEpo) treatment and various anti-hypertensive medications, pre-dialysis arterial blood pressure (BP), prevalence of cardiovascular disease (CVD), and inflammation-related CVD risk factors.
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Patients and methods |
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Within the entire group of 100 patients, there was a subgroup of 70 wherein in whom simultaneous measurements of serum C-reactive protein (CRP), ferritin, and plasma lipoprotein(a) [Lp(a)] were available. Thirty age- and sex-matched healthy volunteers served as controls for determination of sTM concentration.
Approval by our institutional review board for human studies was obtained and all patients and controls gave informed consent.
Methods
The patients were investigated during the morning of midweek dialysis days under fasting conditions. Blood was withdrawn from the arterial outlet of the fistula before onset of dialysis and immediately transferred to an accredited laboratory. Some of the serum and plasma samples were also aliquoted and stored at -40°C until further assay.
Complete blood counts were performed on a K-1000 automated haematology analyser (Sysmex, Kobe, Japan). Routine laboratory methods were used to determine blood and urine urea nitrogen, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total cholesterol, phosphorus, and plasma fibrinogen. Serum CRP was analysed by nephelometry using an analyser and reagents supplied by Orion Diagnostica, Espoo, Finland. The detection limit of CRP was 10 mg/l.
Serum sTM was measured using the enzyme-linked immunosorbent assay (ELISA) and Imubind Thrombomodulin kit (American Diagnostica Inc., Greenwich, CT, USA). The detection limit of sTM was 0.3 ng/ml. Serum ferritin and plasma Lp(a) concentrations were determined using the ELISA kits: Ferritin (Dialab Diagnostic, Wien, Austria) and TintElize Lp(a) (Biopool, Umea, Sweden). The ELISAs were performed in duplicates on a 400 SFC photometer (SLT-Labinstruments, Grödig/Salzburg, Austria), and were calibrated using provided reference samples and standards. For calculations of the results, a computer and a curve-fitting program were used. Their calculated intra- and inter-assay coefficients of variations were <10%.
Serum viral hepatitis markers were detected with AxSYM microparticle ELISA kits manufactured by Abbott, Wiesbaden-Delkenheim, Germany. Anti-HCV antibodies were measured with a HCV version 3.0 assay, and HBV surface antigen with a third generation HBsAg (V2) assay, using an AxSYM analyser (Abbott Laboratories, Abbott Park, IL, USA).
Statistical analyses
The normally distributed data provided by Shapiro-Wilk's W test were expressed as mean ±1 SD. The non-Gaussian data were presented as median (range), and were transformed to log10 to normalize their distribution prior to statistical analysis. The problem of zero values in the sTM assay of controls, which are lost in the log10 transformation, was addressed by adding a value equal to one-third of the detection limit to the results of this assay for all patients and controls. For analysis of CRP, all values <10 mg/l were treated as 9 mg/l. As the RRF and Lp(a) data were highly skewed, other mathematical transformations were also attempted. The square root transformation normalized the distribution of Lp(a), but none of the methods normalized the RRF data.
For inter-group comparisons, Student's t-test for independent samples, 2 test and one-way analysis of variance with post hoc Scheffe's procedure were used when appropriate. Bivariate associations were assessed by Pearson's linear correlation analysis and quasi-Newton bivariate logistic regression analysis. The RRF data were analysed using non-parametric Spearman's correlation test. Stepwise multiple linear regression analysis with both forward selection and backward elimination was employed to evaluate any associations between sTM level as the outcome variable and multiple independent variables. Multivariable analysis of the RRF data was not possible because of the non-normal distribution of this variable.
All P values were two-tailed, and values <0.05 were considered statistically significant. Computations were performed using Statistica 5.0 (StatSoft Inc., Tulsa, OK, USA).
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Results |
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Independent predictors of sTM
The independent variables for the multivariable analysis were selected based on their significant correlation with sTM either by linear or logistic bivariate analysis. They included hepatitis marker status, HD duration, type of heparin and dialysate buffer, use of rHuEpo, and pre-dialysis SBP.
The parameters which were also associated with viral hepatitis seropositivity such as increased ALT (2=16.83, P<0.0001) and ALP (
2=7.13, P=0.008), lower BMI (
2=10.93, P=0.009), decreased platelet count (
2=10.20, P=0.001), and lower Lp(a) (
2=4.70, P=0.030), as well as nPCR and total cholesterol, which were negatively correlated with ALT activity (r=-0.336, P=0.0006 and r=0.408, P<0.0001, respectively), were excluded from the list of independent variables as being intervening variables.
Duration of time on HD was positively associated with sTM levels (r=0.325, P=0.001) as well as with viral hepatitis seropositivity (2=41.37, P<0.0001), use of rHuEpo (
2=5.77, P=0.016) and UFH (vs enoxaparin) (
2=5.95, P=0.015). To clarify whether time on HD could be another intervening variable, we analysed the association between HD its duration and sTM in two groups of patients stratified by hepatitis marker status. There was a significant positive correlation between HD duration and sTM in the patients with hepatitis markers (r=0.306, P=0.022) and a positive association of borderline significance in the group without (r=0.284, P=0.069). Based on these results, we could not definitively either exclude any specific effects of HD duration on sTM levels or confirm an intervening action of this variable. Therefore, HD duration was considered as an independent variable for the multivariable analysis. Although the effects of rHuEpo and UFH on sTM levels could reflect our bias to the preferred use of these medications in long-term dialysis patients, these confounders were included in the multivariable model to determine whether they were independently predictive of sTM levels when adjusted for HD duration.
The ACEI-treated versus untreated patients were more frequently treated with rHuEpo (n=29 (76%) vs n=28 (45%), P=0.003), and there was a positive association between ACEI use and pre-dialysis SBP (2=7.18, P=0.007). Therefore, treatment with ACEI was not included in the multivariable model as being an intervening variable. Pre-dialysis DBP was not included in this analysis because of its multicollinearity with SBP (r=0.830).
The forward stepwise multiple linear regression analysis (Table 3) showed that positive hepatitis markers, use of UFH and rHuEpo, and raised pre-dialysis SBP were significant independent predictors of increased sTM levels in chronic HD patients. The backward analysis as well as the analyses using pre-dialysis DBP instead of SBP yielded the same results (data not shown).
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Discussion |
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The positive viral hepatitis marker was the strongest independent predictor of elevated sTM levels. In support of this phenomenon, the associations between sTM and certain surrogates or consequences of liver dysfunction closely resembled those found between viral hepatitis seropositivity or ALT levels and these variables. Notably, sTM was negatively related to serum ALT, ALP, and liver-synthesized cholesterol and Lp(a). The inverse association found between sTM and nPCR could reflect impaired hepatic synthesis of urea, a more restrictive diet, anorexia and low protein intake, or malabsorption because of intestinal congestion in patients with diseased livers. Malnutrition secondary to liver damage was reflected by the negative relationship between sTM and BMI. The high sTM levels associated with low platelet counts was likely a result of splenic sequestration of platelets in patients with chronic liver disease. These relationships are consistent with the concept that sTM levels in maintenance HD patients are affected by viral infection and/or dysfunction of the liver.
In this and other reports [6,7,11], sTM levels were positively associated with longer duration of HD therapy. However, the issue of viral hepatitis prevalence and liver dysfunction in relation to sTM was not addressed in the previous studies. As could be expected from the natural history of viral hepatitis infection in this population [13], the development of this disease was directly related to increasing time on HD in our patients. Nevertheless, the positive association between HD duration and sTM levels was significant in the patients with hepatitis markers, but not in those without. This information combined with the finding that time on HD was not predictive of sTM levels in the multivariable analysis when adjusted for hepatitis marker status, strongly indicate that increased sTM concentrations reflect higher prevalence of viral hepatitis in long-term HD patients rather than progressive EC stimulation due to repeated HD procedures.
Increases in sTM during the course of viral hepatitis could be explained by defective hepatic degradation and/or enhanced expression and release of this glycoprotein from EC of the diseased liver. Although the former process has not been adequately studied in humans, the liver appears to be the major site of TM clearance in mice [14]. On the other hand, there is good evidence of enhanced TM synthesis by the diseased liver, as demonstrated by recent findings of increased TM expression in hepatic sinusoidal EC in patients with chronic HBV and HCV infection [15], and by the up-regulation of TM in EC and its concomitant release into the blood of animals in the course of acute liver damage [16]. In addition, enhanced TM expression in hepatic EC and high sTM levels in vena cava effluents has been reported in patients with poor liver graft function [17]. In two other studies, activation of sinusoidal EC during viral hepatitis was demonstrated by the finding of increased levels of soluble vascular cell adhesion molecule 1 [18], and by the up-regulation of this selectin in hepatic EC [19].
Thus, it is plausible that the increased levels of sTM in our HD patients with viral hepatitis are due to overexpression of TM in hepatic EC rather than to permanent liver injury. However, decreased hepatic degradation of sTM cannot be definitively excluded in the present study.
Impaired renal function is the most important determinant of sTM levels in pre-dialysis patients [810], and is probably the main cause for its prominent increase during chronic HD. However, in this study, we found no association between sTM and RRF in subjects with preserved renal function. The fact that this association was present in the whole population appears to result from the higher prevalence of viral hepatitis in patients that became anuric during on prolonged HD therapy. These data suggest that the influence of remnant renal excretory function on sTM levels is no longer important in maintenance HD patients, and further support the predictive role of viral liver disease on this marker.
Regarding the relationship between sTM levels and cardiovascular status, this endothelial marker was not associated with CVD prevalence in our HD population. On the other hand, pre-dialysis BP was found to be an independent positive predictor of the sTM levels when adjusted for potential confounders, such as the type of heparin and dialysate buffer used. This is the first report of such an association in HD patients and requires further confirmation. In relevant studies, sTM levels were elevated in hypertensive diabetic patients in comparison with the similar normotensives [3]; however, there was no such BP association in healthy subjects [4]. Regarding markers of inflammation, a process closely connected with the development of CVD [20], sTM was found to be negatively related to Lp(a), but not to CRP, ferritin, or fibrinogen. Lp(a) is a recently recognized independent CVD risk factor in HD patients [20]. This particle contains the apolipoprotein(a) molecule, which has a close structural homology to plasminogen, and therefore may competitively inhibit fibrinolysis. This provides a deleterious connection between inflammation, haemostasis and atherosclerosis. In our study, the negative association between sTM and Lp(a), which causes EC injury, could also be ascribed to liver biosynthetic dysfunction during the development of viral hepatitis.
Both administration of UFH (but not LMWH enoxaparin) as a blood anticoagulant and rHuEpo treatment continued to be significantly, but weakly, associated with high sTM levels in the multivariable model when adjusted for other covariates, including time on HD. However, in view of the absence of relevant clinical data, further studies are needed to evaluate our hypotheses that enoxaparin is more protective for vascular EC than UFH, and that rHuEpo stimulates TM synthesis and/or release during chronic HD.
Our data support previous findings that elevated sTM levels reflect the anticoagulant status rather than the prothrombotic status [4,5]. Thus, high sTM levels were strongly affected by chronic viral hepatitis and progressive liver dysfunction, two states known to predispose to bleeding rather than to thrombotic complications. Prospective studies will be needed to establish whether high sTM levels have cardiovascular consequences in chronic HD patients, and whether pre-dialysis BP, the type of heparin, and rHuEpo are able to modify the serum levels of this marker.
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Acknowledgments |
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Notes |
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References |
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