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Keywords:

  • losartan;
  • nephritic syndrome;
  • proteinuria;
  • prothrombotic state;
  • thrombin generation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Summary. Background: Overt proteinuria is a strong risk factor for thromboembolism, owing to changes in the levels of various coagulation proteins and urinary antithrombin loss. The described coagulation disturbances in these patients are based on outdated studies conducted primarily in the 1970s and 1980s. Whether these coagulation disturbances resolve with antiproteinuric therapy has yet to be studied. Methods: A total of 32 patients with overt proteinuria (median, 3.7 g day−1; interquartile range, 1.5–5.6) were enrolled in this intervention crossover trial designed to assess optimal antiproteinuric therapy with sodium restriction, losartan, and diuretics. Levels of various procoagulant and anticoagulant proteins, and parameters of two thrombin generation assays (calibrated automated thrombogram [CAT] and prothrombin fragment 1 + 2) were compared between the placebo period and the maximum antiproteinuric treatment period. As a secondary analysis, coagulation measurements of the placebo period in these patients were compared with those of 32 age-matched and sex-matched healthy controls. Results: Median proteinuria was significantly lower during the maximum treatment period (median, 0.9 g day−1; interquartile range, 0.6–1.4; P < 0.001) than during the placebo period. Similarly, levels of various liver-synthesized procoagulant and anticoagulant proteins, activated protein C resistance and prothrombin fragment 1 + 2 levels were significantly lower during the maximum treatment period than during the placebo period. However, von Willebrand factor and factor VIII levels were similar. On the basis of the higher levels of procoagulant proteins (fibrinogen, FV, FVIII, and von Willebrand factor) and both thrombin generation assays, patients were substantially more prothrombotic than healthy controls (P < 0.004). Conclusions: Antiproteinuric therapy ameliorates the prothrombotic state. Proteinuric patients are in a more prothrombotic state than healthy controls.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Nephrotic syndrome is a known strong risk factor for both arterial and venous thromboembolism [1–4]. Nephrotic syndrome is characterized by urinary protein losses in excess of 3.5 g per 1.73 m2 body surface area per day in association with hypoalbuminemia, hypercholesterolemia, and peripheral edema. The exact pathophysiologic mechanisms of thromboembolism in patients with nephrotic syndrome have yet to be unraveled. Low levels of antithrombin resulting from urinary loss and alterations in plasma levels of various proteins involved in coagulation are considered to be the main predisposing factors [4–6]. Enhanced platelet aggregation, decreased fibrinolysis, hyperviscosity and hyperlipidemia are other, less often postulated, mechanisms that may be responsible for the prothrombotic state in these patients [4,7–9]. Finally, in the general population, generalized endothelial dysfunction and low-grade inflammation, as reflected by microalbuminuria and elevated levels of high-sensitivity C-reactive protein, respectively, may be additional factors supporting a prothrombotic state in proteinuric subjects [10–12].

Although the association between the extent of proteinuria and changes in plasma levels of various coagulation proteins, including low plasma antithrombin levels, has been considered to be common textbook knowledge, solid evidence is lacking. The previously reported high risk of thromboembolism at presentation of nephrotic syndrome and the positive correlation between the extent of proteinuria and coagulation disturbances or venous thromboembolism suggest that antiproteinuric therapy may reduce the risk of thromboembolism in these patients [1,5,13,14]. It is of note that the supposed beneficial effect of antiproteinuric therapy on the prothrombotic state rests on observational studies with immunosuppressive regimens [5,13,14]. Studies evaluating the impact of intervention in the renin–angiotensin system, which is currently the cornerstone of antiproteinuric treatment, on coagulation disturbances in patients with overt proteinuria have yet to be conducted. Therefore, we set up this study to assess the impact of renin–angiotensin system-inhibiting antiproteinuric therapy on the prothrombotic state. We also assess how the prothrombotic state in proteinuric subjects compares with healthy volunteers.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Patients and study protocol

This study was conducted on patients who participated in a randomized, double-blind, placebo-controlled, crossover trial designed to investigate the effects of sodium restriction, hydrochlorothiazide and their combination on proteinuria during losartan use in patients with overt proteinuria [15]. Details of the study protocol have been published elsewhere [15]. In brief, patients at our outpatient nephrology clinics with overt proteinuria and stable renal function (i.e. creatinine clearance > 30 mL min−1), aged 18–70 years, were considered to be eligible. Patients with uncontrolled hypertension (mean arterial pressure > 100 mmHg), serum potassium > 5.5 mm, cardiovascular disease, contraindication for losartan or hydrochlorothiazide use and/or diabetes were excluded, as were frequent users of non-steroidal anti-inflammatory drugs (> 2 doses week−1). All enrolled patients were treated for 6-week periods with placebo, losartan, and losartan plus hydrochlorothiazide, in combination with either a high-sodium or low-sodium diet, in random order (Fig. 1) [15]. In one of 33 patients, citrate plasma was not available; this patient was therefore excluded from current analysis. For the current study, blood and urine samples from the placebo period were compared with those from the period with the strongest antiproteinuric response, that is, the period with losartan, low sodium, and hydrochlorothiazide. Samples from both the placebo period and maximum treatment period were collected in the last week of the period (i.e. the sixth week). The study was approved by the local medical ethics committee, and was conducted in accordance with the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants.

image

Figure 1.  Flow-chart of the trial. Selected patients entered the randomized, placebo-controlled, crossover study and were consecutively treated for 6 weeks with placebo, losartan (100 mg once daily) and losartan plus hydrochlorothiazide (HCT) (100/25 mg once daily) in random order. In addition, patients were randomized to start with a high-salt diet (200 mmol sodium daily [approximately 4.8 g]) or a low-salt diet (50 mmol sodium daily [approximately 1.2 g]) for 18 weeks (three 6-week periods). After 18 weeks, patients switched diet, and the three 6-week periods were repeated. As can be seen from the flow diagram, there were four randomization options to which patients were assigned. For current analysis, blood and urine samples from the 6-week placebo period were used for placebo period measurements. Blood and urine samples obtained during the period with the largest antiproteinuric effect, that is, the period with losartan + HCT combined with a low-salt diet, were used for the treatment measurements.

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To determine whether proteinuric subjects have altered coagulation status as compared with healthy individuals, we recruited a group of healthy control subjects from employees of the hospital. All patients were matched with control subjects for sex and age within 10 years. Controls were healthy volunteers who had no (family) history of thromboembolism or renal disease, were not pregnant, and had not used oral contraceptives for at least 3 months before testing.

Study measures

Blood samples were obtained in the morning hours between 8:00 and 9:30 during both the placebo and maximum treatment periods. Venous blood samples were anticoagulated with 1 : 10 volume of 0.109 m trisodium citrate. Platelet-poor plasma was prepared by centrifugation at 2500 × g for 15 min, aliquoted, immediately frozen at − 80 °C, and analyzed later after thawing at 37 °C for 15 min.

On the day before study visits, patients collected 24-h urine samples to determine proteinuria and creatinine excretion. Urinary protein was determined with the pyrogallol red–molybdate method. Serum creatinine, cholesterol and albumin levels were determined with an automated multi-analyzer (SMA-C; Technicon, Tarrytown, NY, USA). Quantitative determination of fibrinogen was performed with Siemens Thrombin Reagent (Siemens, Marburg, Germany) on a Sysmex CA-7000 automated coagulation analyzer (Siemens Diagnostics, Leusden, The Netherlands). Factor V:C and FVIII:C levels were assessed with a one-stage clotting assay (Siemens). Total protein S antigen and von Willebrand factor levels were measured with different ELISAs, with reagents obtained from Dako (Glostrup, Denmark). Free protein S antigen levels were assessed by ELISA after precipitation of protein S bound to C4-binding protein with 3.75% PEG 6000. Activities of protein C (Berichrom Protein C; Dade Behring, Liederbach, Germany) and antithrombin (Coatest; Chromogenix, Mölndal, Sweden) were assessed with chromogenic substrate assays. Thrombin generation was measured with the calibrated automated thrombogram (CAT) method, developed by Hemker et al. [16] and commercialized by Thrombinoscope BV (Maastricht, The Netherlands). The CAT assay was performed in triplicate, with a polypropylene round-bottomed microtiter plate (Greiner Bio-one, Stonehouse, UK) containing 20 μL of trigger with 1 pm tissue factor and 4 μm phospholipids (Thrombinoscope BV) in the absence and presence of 1.5 nm soluble thrombomodulin (American Diagnostica, Stamford, CT, USA) that was added to 80 μL of platelet-poor plasma, as previously described. After addition of 20 μL of substrate-calcium reagent, the reaction was monitored in a Fluoroskan Ascent reader (Thermo Labsystems OY, Helsinki, Finland) equipped with a 390/460-nm filter set (excitation/emission) and a dispenser. By use of a software program (Thrombinoscope BV), the fluorescent signal was converted to a thrombin concentration by continuous comparison with the signal generated by a thrombin calibrator that was added to a parallel sample of the test plasma [16]. Then, the thrombin concentration was calculated and displayed over time [16]. Five parameters were derived from the thrombin generation curves. The endogenous thrombin potential (ETP, mm min−1) was calculated from the area under the thrombin generation curve corrected for α-macroglobulin–thrombin activity. The lag time (min) was the time to the start of thrombin formation, and was defined as the time needed to reach one-sixth of the peak thrombin height. Peak thrombin (nm) was the maximum concentration of thrombin formed. Time to peak (min) was the time needed to reach the peak thrombin height, and the velocity index (nmol s−1) was the rate of thrombin formation per second (i.e. velocity index = peak thrombin/[peak time − lag time]). The ETP and peak thrombin were given as percentages relative to human normal pooled platelet-poor plasma assayed in the same run, as described previously [17]. The protein C and protein S anticoagulant pathway was assessed by the addition of thrombomodulin. Relative ETP reduction after thrombomodulin addition was calculated as 100 − (ETP in the presence of thrombomodulin/ETP in the absence of thrombomodulin) × 100%. Relative peak thrombin reduction after thrombomodulin addition was calculated as 100 − (peak thrombin in the presence of thrombomodulin/peak thrombin in the absence of thrombomodulin) × 100%. To assess the magnitude of in vivo thrombin generation, we also measured prothrombin fragment 1 + 2, which is released from prothrombin during activation by FXa, using a commercially available ELISA system (Enzygnost F1 + F2; Dade Behring, Marburg, Germany). Mean arterial pressure was calculated as diastolic pressure plus one-third of the pulse pressure based on the average of four readings.

Laboratory technicians who measured the levels of (anti)coagulant proteins and performed the CAT and prothrombin fragment 1 + 2 assays were not aware of the extent of proteinuria or the demographics of either study participants or controls.

Statistical analysis

The effect of treatment was evaluated with two-level mixed effect linear regression models, with individual participants being nested within the four trial arms. Models were further adjusted for age, sex, creatinine clearance and hematocrit to account for volume depletion and renal function variability. Variables were log-transformed if not normally distributed, and results are presented as adjusted mean during placebo vs. maximum treatment period with 95% confidence intervals (CIs) and P-values.

The associations between coagulation parameters and the extent of proteinuria were evaluated with the placebo period measurements by fitting crude and adjusted linear regression models. Skewed variables were first transformed to a normal distribution prior to fitting of the models, and results are presented as standardized beta with the corresponding P-values.

Continuous variables are presented as medians with interquartile range (IQR). Categorical data are presented as counts and frequencies. For continuous data, differences between patients and the healthy controls were evaluated with the Wilcoxon rank-sum test. A two-tailed P-value of < 0.05 was considered to indicate statistical significance. All statistical analyses were performed with stata software version 11.2 (Stata-Corp LP, College Station, TX, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Among the 32 patients enrolled in the trial, 28 had renal biopsy-proven glomerulopathies, and four cases of renal biopsy were inconclusive. The underlying glomerulopathies were focal segmental glomerular sclerosis (n = 7), membranous glomerulopathy (n = 6), hypertensive nephropathy (n = 5), IgA nephropathy (n = 5), membranoproliferative glomerulonephritis (n = 2), minimal-change disease (n = 2), and Alport syndrome (n = 1). The median age at enrollment was 51 years (IQR 43–59), and 24 (75%) of the patients were male.

Figure 2 shows the extent of proteinuria and levels of serum albumin, cholesterol, high-sensitivity C-reactive protein, various procoagulant and anticoagulant proteins, CAT parameters and prothrombin fragment 1 + 2 during the placebo period and maximum treatment period. As we previously reported, sodium restriction, hydrochlorothiazide and their combination during losartan use reduced the extent of proteinuria and serum cholesterol levels, and increased serum albumin levels (Fig. 2A) [15]. Mean proteinuria adjusted for sex, age, creatinine clearance and hematocrit was reduced from 2.7 g day−1 (95% CI 2.0–3.7) during the placebo period to 0.8 g day−1 (95% CI 0.6–1.0) during the maximum treatment period (P < 0.001) (Fig. 2A). Similarly, mean arterial pressure was reduced from a mean ± standard deviation of 105 ± 15 mmHg during the placebo period to 90 ± 8 mmHg during the maximum treatment period (P < 0.001). After accounting for the study design, age, sex, creatinine clearance, and hematocrit, the mean levels of the procoagulant proteins fibrinogen and FV were reduced during the maximum treatment period as compared with the placebo period (Fig. 2B). Levels of the anticoagulant proteins protein C and free protein S were lower, and total protein S and antithrombin levels were unaltered (Fig. 2C). Interestingly, of the CAT parameters (i.e. ETP and peak thrombin), only peak thrombin in the presence of thrombomodulin was significantly lower during the maximum treatment period than during the placebo period (Fig. 2D). Adjusted mean levels of other CAT parameters (i.e. lag time, time to peak, and velocity index) were also similar during the maximum treatment period and the placebo period (data not shown). Mean levels of prothrombin fragment 1 + 2 were significantly lower during the maximum treatment period (Fig. 2D). Moreover, activated protein C resistance was ameliorated during the maximum treatment period as compared with the placebo period, as assessed by the mean relative reduction in ETP (adjusted P = 0.08) and peak thrombin (adjusted P = 0.002). Using the median proteinuria reduction (i.e. 2.1 g day−1) during the maximum treatment period vs. placebo period, we performed an additional sensitivity analysis to assess the corresponding coagulation changes in the 50% of subjects with the highest proteinuria reduction (i.e. > 2.1 g day−1). Generally, the effects were consistent with the overall associations, except for antithrombin, which showed more pronounced elevation during treatment (adjusted = 0.02) in the 50% of the patients with the best antiproteinuric response (i.e. > 2.1 g day−1).

image

Figure 2.  Components of nephrotic syndrome, high-sensitivity C-reactive protein (hs-CRP), various procoagulant and anticoagulant proteins and functional coagulation assays during the placebo period vs. the maximum treatment period. (A) Nephrotic syndrome parameters and hs-CRP. (B) Procoagulant protein levels. (C) Anticoagulant protein levels. (D) Parameters of calibrated automated thrombogram and prothrombin fragment 1 + 2 (Prothrombin F1+2) levels. Error bars depict the adjusted mean values and 95% confidence intervals. P-values are adjusted for repeated measures, trail arms, age, sex, creatinine clearance, and hematocrit. In (D), the gray error bars represent (relative) peak thrombin and the black error bars depict (relative) ETP. ETP, endogenous thrombin potential; TM, thrombomodulin; VWF, von Willebrand factor; rETP, relative ETP; rPeak, relative peak.

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To assess the general hypothesis that coagulation disturbances are directly associated with the extent of proteinuria, we investigated the association of proteinuria levels with the levels of coagulation proteins and the coagulation functional assays, using the placebo period blood and urine samples (Table 1). Age-adjusted and sex-adjusted associations of proteinuria with both procoagulant and anticoagulant proteins were positive, except for antithrombin, which was not associated with proteinuria at all. After additional adjustment for creatinine clearance, the observed associations were no longer significant, except for fibrinogen. CAT parameters were not related to the extent of proteinuria, in either crude or adjusted analysis. Prothrombin fragment 1 + 2 levels were positively associated with proteinuria in the crude analysis. Finally, it is assumed that the upregulation of coagulation protein synthesis is secondary to a reduction in serum albumin levels in proteinuric patients [8,13,18]. When we substituted proteinuria with serum albumin, the associations presented in Table 1 were, in general, somewhat weaker.

Table 1.   Association between the extent of proteinuria and coagulation disturbances assessed during the placebo period
VariableMolecular mass (kDa)Crude standardized betaP-valueAdjusted standardized beta*P-value*Adjusted standardized beta†P-value†
  1. CAT, calibrated automated thrombogram; ETP, endogenous thrombin potential; NA, not applicable; TM, thrombomodulin. *Adjusted for age and sex. †Adjusted for age, sex, and creatinine clearance.

Procoagulant proteins
 Fibrinogen3400.540.0010.550.0020.480.01
 Factor V3300.470.0070.400.030.260.16
 Factor VIII3300.450.010.430.020.300.10
 von Willebrand factor> 10000.370.040.390.050.250.20
Anticoagulant proteins
 Antithrombin580.200.270.090.660.000.99
 Protein C620.400.020.370.060.200.32
 Protein S total0.240.200.410.040.290.14
 Protein S free690.310.090.39< 0.050.330.10
Tests for prothrombotic state
 CAT parameters
  ETP (%)NA0.070.690.120.560.120.58
   With TMNA− 0.040.81− 0.001.00.040.84
  Lag time (min)NA0.180.340.340.080.240.23
   With TMNA0.140.450.290.150.170.40
  Peak thrombin (%)NA− 0.050.79− 0.030.88− 0.000.98
   With TMNA− 0.100.59− 0.100.64− 0.040.84
  Time to peak (min)NA0.160.390.290.140.210.30
   With TMNA0.170.350.320.110.220.28
  Velocity index (nmol s−1)NA− 0.040.82− 0.060.76− 0.030.89
   With TMNA− 0.100.58− 0.130.53− 0.080.71
  Prothrombin fragment 1 + 20.380.040.330.100.230.27

To assess how the prothrombotic state in proteinuric subjects compares with healthy individuals, we compared coagulation parameters of the patients during the placebo period with those of 32 age-matched and sex-matched healthy control subjects (Table 2). The levels of procoagulant fibrinogen, FV, FVIII and von Willebrand factor were significantly higher in patients than in controls (P ≤ 0.004). In contrast, the levels of anticoagulant antithrombin, protein C and total protein S were similar in patients and controls (P ≥ 0.31). However, the levels of free protein S were significantly higher in patients than in controls (P < 0.001). Both the CAT and the prothrombin fragment 1 + 2 assay demonstrated a more procoagulant state in patients than in controls.

Table 2.   Comparison of patients’ measurements during the placebo period with healthy controls
DemographicsPatientsControlsP-value
  1. CAT, calibrated automated thrombogram; ETP, endogenous thrombin potential; TM, thrombomodulin. *Values are medians and interquartile ranges.

Subjects, n3232
 Male, n (%)24 (75)24 (75)
Age (years)*51 (43–59)46 (32–52)
Laboratory measurements*
 Fibrinogen (g L−1)3.9 (3.6–4.3)2.7 (2.4–2.9)< 0.001
 Factor V (%)137 (111–164)104 (83–121)0.004
 Factor VIII (%)180 (140–213)117 (94–136)< 0.001
 von Willebrand  factor (%)191 (151–232)100 (85–114)< 0.001
 Antithrombin (%)98 (93–109)99 (93–108)0.70
 Protein C (%)113 (103–131)109 (100–126)0.51
 Total protein S (%)129 (111–140)121 (104–135)0.31
 Free protein S (%)127 (101–147)81 (66–101)< 0.001
CAT parameters
 ETP (%)163 (146–191)96 (76–110)< 0.001
  With TM147 (126–171)44 (33–55)< 0.001
 Lag time (min)3.6 (3.2–4.3)3.3 (3.0–3.8)0.24
  With TM3.6 (3.1–4.3)3.1 (3.0–3.7)0.18
 Peak thrombin (%)280 (242–328)90 (66–123)< 0.001
  With TM242 (203–294)64 (49–84)< 0.001
 Time to peak (min)6.8 (5.9–7.3)8.3 (7.4–9.0)< 0.001
  With TM6.6 (5.9–7.0)6.7 (6.3–7.3)0.16
 Velocity index  (nmol s−1)80 (66–113)19 (14–36)< 0.001
  With TM85 (61–121)22 (14–35)< 0.001
 Relative ETP (%)10 (6–14)52 (43–59)< 0.001
 Relative peak  thrombin (%)15 (8–18)24 (15–32)0.001
 Prothrombin fragment   1 + 2 (pm)330 (209–419)158 (123–197)< 0.001

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

This study explored the effect of renin–angiotensin system-inhibiting antiproteinuric therapy on coagulation disturbances. Antiproteinuric therapy reversed the prothrombotic state, as reflected by a reduction in prothrombin fragment 1 + 2, amelioration of activated protein C resistance, and reductions in various hepatocyte-synthesized coagulation factors, as well as an antithrombin increase, in patients with more than 2.1 g day−1 proteinuria reduction. Except for antithrombin levels, which had no association with the extent of proteinuria, age-adjusted and sex-adjusted levels of all other measured procoagulant and anticoagulant proteins were positively correlated with the extent of proteinuria. Patients were more prothrombotic than healthy controls, as demonstrated by markedly elevated procoagulant protein levels and the functional coagulation assays (i.e. CAT and prothrombin fragment 1 + 2).

This is the first study addressing the effect of renin–angiotensin system-inhibiting antiproteinuric therapy on coagulation disturbances in patients with overt proteinuria. Antiproteinuric therapy resulted in decreased levels of predominantly hepatocyte-synthesized coagulation proteins, that is, fibrinogen, FV, protein C, and free protein S. Conversely, antithrombin levels were significantly increased in patients with proteinuria reduction of more than 2.1 g day−1. In previous studies, mainly in children with nephrotic syndrome, remission of nephrotic syndrome with corticosteroid or immunosuppressive therapy reversed the especially high fibrinogen levels and low antithrombin levels [5,13,14]. The lack of decreases in FVIII, von Willebrand factor and total protein S levels might be explained by persistent endothelial damage and/or inflammation that, to some extent, might be supported by unchanged elevated levels of high-sensitivity C-reactive protein.

Of the functional assays, prothrombin fragment 1 + 2 levels, which reflect in vivo thrombin generation, were significantly reduced during antiproteinuric therapy as compared with the placebo period. Whereas the CAT assay [16] clearly demonstrated a higher procoagulant state in patients than in healthy controls (Table 2), no effect of antiproteinuric treatment on the overall procoagulant state was found. However, the CAT assay did show amelioration of activated protein C resistance (i.e. relative ETP and relative peak thrombin reduction) during treatment as compared with the placebo period. Although the amelioration in activated protein C resistance in our study is not reflected by a reduction in FVIII levels, previous studies have linked high levels of FVIII to activated protein C resistance [17,19]. Moreover, in hereditary thrombophilia with single defects, the CAT assay is recommended as the method of choice for assessment of overall procoagulant state [20,21].

The associations of coagulation proteins levels with proteinuria, without adjustment for creatinine clearance (Table 1), are comparable to the findings of previous studies conducted in patients with untreated nephrotic syndrome, which mainly date back to the 1970s and 1980s [2,6,8,13,14]. It could be debated whether adjustment for creatinine clearance is justified in these patients with primary glomerulopathies, where glomerular damage is the cause of both glomerular filtration rate deterioration and proteinuria. On the basis of previous studies, it is generally accepted that the prothrombotic state in nephrotic patients is attributable to elevated levels of various procoagulant proteins and low antithrombin levels [2,6,8,13,14]. Low plasma antithrombin levels are presumed to be caused by urinary loss [4,5]. We observed normal circulating levels of antithrombin in our proteinuric patients, whereas most previous studies in proteinuric patients found decreased levels [4]. This might be explained by differences in the extent of proteinuria, the underlying glomerulopathies, or the age of the patients, as low level of serum antithrombin are more frequently reported in children [4,5]. Finally, in a small pilot study, we could confirm the presence of considerable amounts of antithrombin (6–21%) in the urine of three patients with proteinuria ranging from 0.5 to 1.5 g day−1 (data not shown). This finding is in line with previous reports [4,22,23], and may explain the lack of association of plasma antithrombin levels with the extent of proteinuria, as upregulation of antithrombin synthesis was probably counterbalanced by urinary loss. This notion of a significant amount of antithrombin loss in the urine is further supported by the fact that patients treated for proteinuria showed an increase in serum antithrombin, especially those with proteinuria reduction of > 2.1 g day−1.

Finally, one can argue that the observed amelioration of the coagulation disturbance may be a direct effect of renin–angiotensin system inhibition by losartan or an altered volume status by diuretics (HCT) rather than proteinuria reduction. However, on the basis of the literature [8,13,22] and our creatinine clearance-adjusted and hematocrit-adjusted results, it is likely that the observed amelioration of coagulation disturbances is attributable to the proteinuria reduction.

This study has several limitations. First, we measured only a selection of coagulation proteins. However, this selection was performed with a predefined aim by assessing the roles of antithrombin, the protein C and protein S pathway, endothelial damage, liver synthesis function, and thrombin generation. Second, the association between coagulation disturbances caused by proteinuria might be better demonstrated in more extreme proteinuria; in our study, almost half of the patients had proteinuria of < 3.5 g day−1. Obviously, more extreme phenotypes of overt proteinuria are rare nowadays, since the availability of effective antiproteinuric medication. Absence of proteinuria in the control group was not ascertained by urine examination. Nevertheless, given that none of the control subjects reported any history of renal disease, and given the low prevalence of overt proteinuria in healthy individuals, it is unlikely that any of our control subjects had overt proteinuria. Moreover, although the age match criteria of 10 years may be large, the true observed median age difference between patients and controls was only 5 years, and additional age adjustment had no influence on coagulation protein levels and coagulation assays in the comparison of patients with controls (data not shown).

In conclusion, antiproteinuric therapy ameliorates the prothrombotic state. Proteinuric patients are in a more prothrombotic state than healthy controls.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

B. K. Mahmoodi had full access to all of the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis; B. K. Mahmoodi, F. Waanders, A. B. Mulder, G. J. Navis, and H. C. Kluin-Nelemans: took part in the study design; F. Waanders, M. C. J. Slagman, L. Vogt and R. Mulder: responsible for data collection; B. K. Mahmoodi analyzed the data, and drafted the manuscript. All authors participated in the interpretation and presentation of the results. All authors have read and approved the final version.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

We thank W. van der Schaaf and K. S. M. Hegge-Paping for their technical assistance.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

H. ten Cate received research grants from the Center for Translational Molecular Medicine and the Netherlands Heart Foundation, and he has received honoraria as a consultant to Boehringer Ingelheim GmbH. The other authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  • 1
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