TR-06011; No of Pages 6 Thrombosis Research xxx (2015) xxx–xxx
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Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography Meng-jie Huang, Ri-bao Wei ⁎, Zi-cheng Wang, Yue Xing, Yu-wei Gao, Min-xia Li, Guang-yan Cai, Xiang-mei Chen Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
a r t i c l e
i n f o
Article history: Received 7 December 2014 Received in revised form 24 May 2015 Accepted 30 June 2015 Available online xxxx Keywords: nephrotic syndrome membranous nephropathy minimal change disease thromboelastography hypercoagulability
a b s t r a c t Introduction: Thromboelastography (TEG) was performed to assess potential hypercoagulability in Nephrotic syndrome (NS) patients with membranous nephropathy (MN) and to explore correlated factors contributing to hypercoagulable status Materials and Methods: 101 MN patients, 61 minimal change disease (MCD) patients and 20 healthy controls met the inclusion criteria. The MN and MCD patients were stratified into two layers according to serum albumin (SALB) levels (b20 g/l or 20–30 g/l). Primary outcome measures included reaction time (R), α-angle, maximum amplitude (MA) and coagulation index (CI). TEG parameters of four patient subgroups were analyzed in factorial designed ANOVA with factors disease and SALB. Results: By linear regression analysis, TEG parameters in MN patients correlated with SALB (P b 0.01) and the ANOVA for factorial designed data confirmed that the main effects of factors SALB and disease were both statistically significant. Besides, comparison between control group and patient subgroups showed that R value in normal controls was significantly higher than that in MN subgroups, but was not statistically different from that in MCD subgroups. NS patients (MCD, MN) had significantly higherα-angle, MA and CI values than healthy controls (p b 0.05). Conclusions: MN patients tend to be more hypercoagulable than normal and MCD patients. Hypercoagulability in MN patients involves the whole thrombotic processes acceleration (activated intrinsic pathway, fibrinogen, platelet function and fibrin-platelet interaction), whereas hypercoagulable state in MCD patients may be that the coagulation factors are not fully activated. Greater efforts should be made to prevent hypercoagulability especially for MN patients with severe hypoalbuminemia. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nephrotic syndrome (NS) patients are prone to thromboembolic events. It is presently thought that the mechanisms are possibly associated with the involvement of multiple factors in the blood coagulation pathway [1–3]. Membranous nephropathy (MN) is the pathological type of NS associated with the highest incidence of thromboembolic events [2–4]. Hypercoagulability is defined as abnormalities in any of the steps that promote coagulation and is important part of early diagnosis of thromboembolic disease. Compared with other pathological types, the difference and the underlying mechanisms of MN-related hypercoagulability are not fully understood. Thromboelastography (TEG) is a blood coagulation assay that can dynamically monitor the entire blood coagulation cascade. Thus far, TEG has been widely used ⁎ Corresponding author at: Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China. E-mail address: [email protected] (R. Wei).
in various clinical fields, such as cardiovascular surgery, trauma, and oncology [5,6]. However, no controlled studies have been reported on the mechanisms of hypercoagulability in NS. Therefore, we retrospectively evaluated the records of TEG in NS patients including the pathological types MN and minimal change disease (MCD). The mechanisms underlying the hypercoagulable state in NS patients with two different pathological types were explored, and the relevant factors influencing the changes in coagulation function in MN patients were analyzed. 2. Material and methods 2.1. Study population This study retrospectively involved inpatients with NS who both had undergone renal biopsy with the diagnosis of MN and MCD in our hospital and had taken TEG examination from 2012 to 2014. Inclusion criteria: (1) urinary protein ≥ 3.5 g/24 h and serum albumin b30 g/L; (2) aged 18 to 70 years; (3) normal liver function; and (4) no previous history of thrombosis or hemorrhagic disease. Exclusion criteria:
http://dx.doi.org/10.1016/j.thromres.2015.06.031 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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(1) secondary renal disease; (2) presence of risk factor(s) for hypercoagulable state, such as the acute phase of infection, recent trauma, surgery, or cancer, and pregnancy; (3) administration of hormones, anticoagulants, or diuretics within one month; (4) treatment with plasma or albumin infusion, plasma exchange, or hemodialysis. A total of 116 MCD patients and 171MN patients received TEG examination. There were 12 MCD patients and 43 MN patients who did not meet the diagnostic criteria for NS, 31 MCD patients and 25 MN patients receiving hormones or anticoagulant therapy prior to admission, 12 MCD patients aged b18 years, and 2 MN patients with abnormal liver function who were excluded from the study. In total, 61 NS patients with MCD and 101 NS patients with MN were included, while 20 healthy adults volunteered as normal controls. The MN and MCD patients were stratified into two layers according to serum albumin levels (b20 g/l or 20–30 g/l). There were 32 MN patients and 27 MCD patients in SALB b20 g/l level, whereas 69 MN patients and 34 MCD patients in SALB 20–30 g/l level. All renal-biopsy-patients had signed the informed consent of Renal Clinical Database Establishment when hospitalized, allowing their data for clinical research. A description of the study flow is depicted in Fig. 1. 2.2. Physical and Clinical data We collected physical and clinical data from101 MN patients and 61MCD patients. These data included Body height, body mass, systolic blood pressure (SBP), diastolic blood pressure (DBP), hemoglobin (Hbg), platelet count(PLT), liver function, serum total protein, serum albumin (SALB), serum creatinine (Scr), cholesterol(CH), triglycerides(TG), serum IgG and 24-h urine protein excretion (UPr). Body mass index (BMI) was calculated in formulae as follows: BMI = weight (kg) / [height (m)]2. Mean arterial pressure (MAP) was calculated in formulae as follows: MAP = (SBP (mmHg) + DBP (mmHg) × 2) /3. The estimated glomerular filtration rate (eGFR) was calculated according to the Modification of Diet in Renal Disease (MDRD) formula: for males; eGFR ml=min=1:73 m2 −1:154 ¼ 186 ðserum creatinineðμmol =LÞ=88: 4Þ ageðyearsÞ−0:203 ; for females; eGFR ml=min=1:73 m2 −1:154 ¼ 186 ðserum creatinineðμmol=LÞ=88: 4Þ
ageðyearsÞ−0:203 0:742:
2.3. Routine coagulation parameters Activated partial thromboplastin time (APTT) and prothrombin time (PT).
(3) maximum amplitude (MA)- indicative of the strength of clot that reflects the cross interaction between platelet functions and coagulation; (4)coagulation index (CI), which represents the overall coagulation profile and is calculated from the R, K, α-angle, and MA values. The typical TEG profile for hypercoagulability is characterized by short R time and highα-angle, MA, CI [7]. 2.5. Statistical analysis Data analysis was performed using Statistical Package for Social Science (SPSS) 17 statistical software. Normally distributed continuous variables were expressed as mean ± standard deviation and nonnormally distributed variables were expressed as median (interquartile range). For continuous data, statistical analysis in patient groups and healthy control group was made by t-test, Mann–Whitney U test, Kruskal-Wallis test or ANOVA as appropriate depending on the normality and levels of the outcome variable. TEG parameters: CI, R, MA and αangle values of four patient subgroups were analyzed in factorial designed ANOVA with factors disease (two levels: MCD, MN) and SALB (two levels: b 20 g/l, 20–30 g/l). Interaction between disease and SALB was included. Categorical variables were compared using the using the χ2 test. Linear regression analysis was used to detect any relation between TEG parameters and clinical data. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Clinical features of 101 NS patients with pathological MN Data of 101 NS patients with pathological MN on age, gender, BMI, MAP, Hbg, PLT, SALB, Scr, BUN, UA, eGFR, TC, TG, UPr, IgG, C3 and Renal pathology were presented in Table 1. 3.2. Linear regression analysis between TEG parameters and clinical variables presented in Table 1 in 101 MN patients To identify the correlated factors influencing TEG parameters (R, α-angle, MA and CI) in MN patients, linear regression analysis was performed on the variables presented in Table 1. The results showed that the TEG parameters of MN patients were mainly correlated with serum albumin. As shown in Fig. 2, CI, α-angle and MA were inversely correlated with serum albumin level (r = − 0.628, P b 0.001; r = − 0.552, P b 0.001; r = − 0.369, P b 0.001). R was positively correlated with serum albumin level (r = 0.432, P b 0.001). Besides, we also found CI was positively correlated with 24-hour urine protein excretion(r = 0.378, P b 0.001) in MN patients. No significant correlation was observed between TEG parameters and age, BMI, MAP, MN stage, or levels of Hbg, PLT, CH, TG, serum IgG, or C3. 3.3. Subject Characteristics
2.4. TEG analysis Coagulation status was assessed via TEG using citrated whole-blood samples (2.0 ml sample volume per tube; 3.8% citrate in 9:1 blood to citrate ratio). For each TEG assay, citrated whole blood (1 ml) was pipetted into to a vial containing 1% kaolin, which was inverted 5 times to ensure mixing of kaolin with the blood. Then, 340 μl kaolin-activated citrated whole blood was transferred to the TEG cup to which 20 μl of 0.2 mol/l CaCl2 had been preloaded for recalcification. The TEG analyzer was stopped 40–60 minutes after reaching maximum amplitude at 37 °C. Parameters included the following: (1) reaction time (R) - time from the start of the test to a TEG amplitude of 2 mm, reflecting the combined effect of coagulation factors involved in the initiation of hemostasis; (2) α-angle- the angle between the tangent line (drawn from the split point to the curve) and the horizontal base line, representing the acceleration of fibrin build-up and cross-linking;
Study above showed that hypoalbuminemia is a risk factor for hypercoagulability in MN patients. We divided MN and MCD patients into two subclasses by the levels of SALB. Table 2 presents detailed stratified data of MN and MCD patients in different subgroups based on SALB (b20 g/l or 20–30 g/l) and clinical features of heathy volunteers. NS patients had significantly higher BMI, MAP, CH, TG and lower SALB, eGFR than healthy controls. MN and MCD patients showed no significant difference in SALB and UPr at the same SALB level. Besides,the whole study population showed no significant differences in age, gender and PLT (P N 0.05). 3.4. Routine coagulation test parameters The five groups showed no significant difference in PT value. In terms of APTT value, there was no significant difference among low
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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Fig. 1. The flow chart of this study.
level SALB MCD group (SALB b 20 g/l), high level SALB MCD group (SALB 20–30 g/l) and normal control group, but the values of these three groups are higher than that of the MN subgroups (P b 0.05). (Table 3) 3.5. TEG parameters Table 4 compares TEG parameters between control group and patient groups. R value in normal controls was significantly higher than that in MN subgroups, but was not statistically different from that in MCD subgroups. NS patients (both MCD and MN) had significantly higherα-angle, MA and CI values than healthy controls(p b 0.05). Besides, CI, R, MA and α-angle values of four NS patient subgroups (Table 4) were analyzed in factorial designed ANOVA with factors
Table 1 Clinical features of 101 NS patients with pathological MN. Clinical characteristics
Patients (n = 101)
Age (y) Gender(male/female) BMI (kg/ m2) MAP(mmHg) Hbg (g/l) PLT (109/l) SALB (g/l) Scr (umol/l) BUN (mg/dl) UA(umol/l) eGFR (ml/min/1.73 m2) TG (mmol/l) TC (mmol/l) UPr (g/day) IgG(mg/dl) C3(mg/dl) Renal pathology I I–II II III
44.94 ± 16.50 60/41 25.12 ± 4.04 96.41 ± 13.40 129.92 ± 19.22 258.25 ± 72.49 22.96 ± 3.93 67.80(57.90, 84.75) 4.79(3.65,6.18) 323.97 ± 102.59 104.64 ± 32.52 1.95(1.42, 2.75) 6.73(5.58, 8.29) 5.75 ± 2.42 474.5(323.3,672.7) 115.46 ± 27.55 55 23 19 4
Data are expressed as mean ± standard deviation (SD) or median (interquatile range) as appropriate; BMI = body mass index; MAP = Mean arterial pressure; Hbg = hemoglobin; SALB = serum albumin; CH = cholesterol; TG = triglyceride; Scr = serum creatinine; BUN = urea nitrogen; UA = uric acid; eGFR = estimated glomerular filtration rate; and UPr = 24-h urine protein excretion.
disease (two levels) and SALB (two levels). Interaction between disease (MCD, MN) and SALB (b 20 g/l, 20–30 g/l) was presented in Fig. 3. The main effect of factor disease was statistically significant (CI value: F = 32.08, P b 0.001; R value: F = 19.24, P b 0.001; MA value: F = 8.31, P = 0.005;α-angle: F = 15.15, P b 0.001) and the main effect of factor SALB was also statistically significant (CI value: F = 31.98, P b 0.001; R value: F = 14.84, P b 0.001; MA value: F = 9.28, P = 0.003;α-angle: F = 17.18, P b 0.001).But no statistically significant interaction between the two factors was found (CI value: F = 0.65, P = 0.42; R value: F = 1.30, P = 0.25; MA value: F = 0.07, P = 0.78;α-angle: F = 1.08, P = 0.30). 4. Discussion Thromboembolic events are one of the most severe complications in patients with NS. Real-time control of the coagulation status of the patient and preventions of the occurrence of thrombotic events are challenging to clinicians. Because of the complexity of NS and the limitations of coagulation test methods, the mechanisms of hypercoagulability in NS patients have not been fully elucidated. TEG serves as an approach to test coagulation that can help assess the coagulation status of the patient. To date, TEG has been clinically used for several clinical settings to evaluate and fix the coagulopathy and to study clotting mechanism. The typical TEG profile for hypercoagulability is defined by the presence of the following: shortened R time, increasedα-angle and MA. In this study, NS patients in the MCD and MN groups are characterized by shorter R times and higherα-angle, MA, CI than healthy adults, indicating that NS patients tended to be hypercoagulable, with higher severity in cases of MN. This finding is consistent with previous reports that MN is the pathological type of NS with the highest incidence thromboembolic events [2,3]. R value mainly reflects the activity of enzymatic coagulation cascade and APTT, PT represent intrinsic and extrinsic pathway respectively. This study found no difference in the R value between MCD patients and healthy controls. However, R value was significantly reduced in the MN subgroups, indicating accelerated enzymatic activity in MN patients. Combined with routine coagulation test, PT value showed no difference among MCD, MN and healthy controls whereas APTT had a consistent variation with R value, indicating hypercoagulability process does not activate extrinsic coagulation pathway but intrinsic pathway.α-angle represents velocity of clot formation
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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Fig. 2. Correlation of serum albumin with TEG parameters in the MN patients. A: Correlation of serum albumin with CI value. B: Correlation of serum albumin with R value. C: Correlation of serum albumin with α-angle value. D: Correlation of serum albumin with MA value.
and increase inα-angle may be due to fibrinogen elevation. MA mainly reflects platelet function and is influenced by the quantity and quality of platelets. The data showed all study population have no significant difference in platelet counts, whereasα-angle was significantly increased in MN and MCD patients and the MA value increased in the order of MN N MCD N healthy control group. The results indicate that increased fibrinogen, abnormal platelet activation and accelerated fibrin-platelet
interaction in MN and MCD patients, especially the former group. Thus, our findings suggest that the hypercoagulable state in MCD patients may be that the coagulation factors preceding the initial fibrin platelet interaction were not fully activated, whereas the hypercoagulable state in MN patients involves the whole thrombotic processes acceleration (accelerated intrinsic pathway, abnormal platelet activation and accelerated fibrin-platelet interaction). To date, studies on the roles of
Table 2 Clinical parameters of MN, MCD patients in different SALB levels and healthy controlsAbbreviations as in Table 1. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
N Age(year) Gender(M/F) BMI(kg/m2) MAP(mmHg) Hbg(g/L) PLT(10^9/L) SALB (g/L) TG(mmol/l) CH(mmol/l) Scr(umol/l) eGFR (ml/min/1.73 m2) UPr(g/day)
20 37.3 ± 13.8 11/9 21.6 ± 2.9 86.9 ± 7.6 137.1 ± 14.0 253.3 ± 53.7 44.4 ± 2.8 1.1 ± 0.5 3.8 ± 0.8 62.7 ± 8.6 121.4 ± 12.8
27 41.2 ± 16.5 16/11 25.2 ± 3.2★ 93.8 ± 10.4★ 135.6 ± 18.2◆ 264.1 ± 57.1 17.1 ± 1.9★# 2.2(1.7, 2.9)★◆ 9.9(7.6, 11.4)★ 79.5(60.7, 96.4)★ 89.3 ± 28.1★#
32 43.6 ± 18.7 19/13 25.6 ± 4.8★ 98.2 ± 13.7★ 125.1 ± 17.3★ 273.7 ± 64.7 18.2 ± 1.9★※ 2.0(1.6, 2.5)★ 7.6(5.8, 9.3)★ 69.6(60.1, 85.3)★ 99.7 ± 33.6★
34 35.9 ± 13.1 21/13 25.2 ± 4.1★ 92.1 ± 14.2★ 138.1 ± 17.1 248.8 ± 69.0 24.1 ± 2.8★ 2.3(1.8, 3.3)★▲ 8.7(7.1, 10.1)★ 67.9(52.8, 85.6)★ 111.1 ± 36.8★
69 45.5 ± 15.3 41/28 24.9 ± 4.5★ 95.3 ± 13.2★ 132.3 ± 19.6 250.1 ± 75.0 25.2 ± 2.3★ 1.9(1.3, 2.9)★ 6.6(5.3, 8.0)★ 65.0(57.1, 84.7)★ 106.5 ± 31.8★
---
6.3(4.7, 8.4)
7.4(6.2, 8.9)※
5.7(3.6,7.7)
4.3(3.7, 5.34)
★ ◆ ▲ ※
#
P b 0.05, vs control group; P b 0.05, MCD with SALB b20 g/l group vs MN with SALB b20 g/l group; P b 0.05, MCD with SALB 20–30 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MN with SALB b20 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MCD with SALB b20 g/l group vs MCD with SALB 20–30 g/l group.
Table 3 Comparison of Routine coagulation test parameters in study population. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
APTT(s) PT(s)
39.7 ± 2.0 12.8 ± 0.40
39.6 ± 3.3◆ 12.8 ± 0.7
34.8 ± 4.9★ 12.7 ± 0.9
37.0 ± 5.4▲ 12.4 ± 0.7
34.7 ± 7.5★ 12.3 ± 0.6
※
P b 0.05, MN with SALB b20 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MCD with SALB b20 g/l group vs MCD with SALB 20–30 g/l group. ★ P b 0.05, vs control group; ◆ P b 0.05, MCD with SALB b20 g/l group vs MN with SALB b20 g/l group; ▲ P b 0.05, MCD with SALB 20–30 g/l group vs MN with SALB 20–30 g/l group;
#
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
M. Huang et al. / Thrombosis Research xxx (2015) xxx–xxx
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Table 4 Comparison of TEG parameters between control group and patient groups. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
R (min) α-angle (deg) MA (mm) CI
7.3 ± 1.1 57.7 ± 5.6 56.6 ± 4.9 −1.9 ± 1.7
6.7 ± 1.0 68.9 ± 5.1★ 71.1 ± 5.4★ 1.24 ± 1.3★
5.5 ± 0.8★ 74.8 ± 3.2★ 73.3 ± 4.8★ 2.8 ± 1.1★
7.2 ± 1.5 65.5 ± 6.6★ 68.1 ± 5.0★ 0.1 ± 1.98★
6.6 ± 1.2★ 68.9 ± 6.2★ 70.9 ± 5.4★ 1.24 ± 1.2★
★
P b 0.05, vs control group.
platelets, fibrinogen and coagulation factors in the hypercoagulability of NS patients have been conducted independently [8–15], limiting our understanding on the comprehensive assessment of coagulation. The present study was the first to use TEG analysis to investigate the effects of global hemostasis assay on the mechanisms of hypercoagulability in patients with different pathological types of NS. The linear regression analysis for hypercoagulability in MN patients showed that hypoalbuminemia was the major risk factor for thrombophilia of the nephrotic syndrome, and further TEG parameters of four NS patient subgroups analyzed in factorial designed ANOVA confirmed that hypercoagulable state of MN patients was accompanied by a decrease in serum albumin. This result confirms previous findings in the literature [15] and suggests a need to strengthen efforts to prevent hypercoagulable state, especially for MN patients with severe hypoalbuminemia. Because of the relatively low correlation coefficient between CI and 24-hour urinary protein excretion, we could not clearly demonstrate urine protein as a risk factor for hypercoagulability [16]. Our study has some potential limitations. Due to its retrospective study design, samples may not well present patient population. We
enrolled MN and MCD patients who had taken TEG examination. As patients who had high clinical suspicion of hypercoagulability might easily undergo TEG examination, our findings may overestimate the degree of hypercoagulable state. Besides, some information for potential hypercoagulability risk factors such as medication history of hormonal contraceptives was not able to obtain. The result of our study is the assessment of potential risk for hypercoagulability by TEG in NS patients with MN. We did not follow up and assess the clinical incidence of thrombosis in the observed patients. Thus the study is not sufficient to use TEG to resolve potential risk for thrombosis in MN patients. This study documents that patients with MN tend to be more hypercoagulable than normal or patients with MCD. The hypercoagulable state in MN patients involves the whole thrombotic processes acceleration (activated intrinsic pathway,increased fibrinogen, abnormal platelet activation and accelerated fibrin-platelet interaction), whereas hypercoagulable state in MCD patients may be that the coagulation factors preceding the initial fibrin platelet interaction are not fully activated. Thus, this study based on TEG analysis has implications for guiding clinical interventions for hypercoagulability in NS. Concomitant
Fig. 3. Main effect and interaction between two factors: disease and SALB. A: Effect of disease and SALB on CI value. B: Effect of disease and SALB on R value. C: Effect of disease and SALB on MA value. D: Effect of disease and SALB on Angle value.
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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anticoagulant and antiplatelet therapy is required for MN patients, while antiplatelet therapy should be emphasized for MCD patients. Since hypercoagulable state is accompanied by a decrease in serum albumin, greater efforts should be made for MN patients with severe hypoalbuminemia.
[9]
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Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography Meng-jie Huang, Ri-bao Wei ⁎, Zi-cheng Wang, Yue Xing, Yu-wei Gao, Min-xia Li, Guang-yan Cai, Xiang-mei Chen Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
a r t i c l e
i n f o
Article history: Received 7 December 2014 Received in revised form 24 May 2015 Accepted 30 June 2015 Available online xxxx Keywords: nephrotic syndrome membranous nephropathy minimal change disease thromboelastography hypercoagulability
a b s t r a c t Introduction: Thromboelastography (TEG) was performed to assess potential hypercoagulability in Nephrotic syndrome (NS) patients with membranous nephropathy (MN) and to explore correlated factors contributing to hypercoagulable status Materials and Methods: 101 MN patients, 61 minimal change disease (MCD) patients and 20 healthy controls met the inclusion criteria. The MN and MCD patients were stratified into two layers according to serum albumin (SALB) levels (b20 g/l or 20–30 g/l). Primary outcome measures included reaction time (R), α-angle, maximum amplitude (MA) and coagulation index (CI). TEG parameters of four patient subgroups were analyzed in factorial designed ANOVA with factors disease and SALB. Results: By linear regression analysis, TEG parameters in MN patients correlated with SALB (P b 0.01) and the ANOVA for factorial designed data confirmed that the main effects of factors SALB and disease were both statistically significant. Besides, comparison between control group and patient subgroups showed that R value in normal controls was significantly higher than that in MN subgroups, but was not statistically different from that in MCD subgroups. NS patients (MCD, MN) had significantly higherα-angle, MA and CI values than healthy controls (p b 0.05). Conclusions: MN patients tend to be more hypercoagulable than normal and MCD patients. Hypercoagulability in MN patients involves the whole thrombotic processes acceleration (activated intrinsic pathway, fibrinogen, platelet function and fibrin-platelet interaction), whereas hypercoagulable state in MCD patients may be that the coagulation factors are not fully activated. Greater efforts should be made to prevent hypercoagulability especially for MN patients with severe hypoalbuminemia. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nephrotic syndrome (NS) patients are prone to thromboembolic events. It is presently thought that the mechanisms are possibly associated with the involvement of multiple factors in the blood coagulation pathway [1–3]. Membranous nephropathy (MN) is the pathological type of NS associated with the highest incidence of thromboembolic events [2–4]. Hypercoagulability is defined as abnormalities in any of the steps that promote coagulation and is important part of early diagnosis of thromboembolic disease. Compared with other pathological types, the difference and the underlying mechanisms of MN-related hypercoagulability are not fully understood. Thromboelastography (TEG) is a blood coagulation assay that can dynamically monitor the entire blood coagulation cascade. Thus far, TEG has been widely used ⁎ Corresponding author at: Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China. E-mail address: [email protected] (R. Wei).
in various clinical fields, such as cardiovascular surgery, trauma, and oncology [5,6]. However, no controlled studies have been reported on the mechanisms of hypercoagulability in NS. Therefore, we retrospectively evaluated the records of TEG in NS patients including the pathological types MN and minimal change disease (MCD). The mechanisms underlying the hypercoagulable state in NS patients with two different pathological types were explored, and the relevant factors influencing the changes in coagulation function in MN patients were analyzed. 2. Material and methods 2.1. Study population This study retrospectively involved inpatients with NS who both had undergone renal biopsy with the diagnosis of MN and MCD in our hospital and had taken TEG examination from 2012 to 2014. Inclusion criteria: (1) urinary protein ≥ 3.5 g/24 h and serum albumin b30 g/L; (2) aged 18 to 70 years; (3) normal liver function; and (4) no previous history of thrombosis or hemorrhagic disease. Exclusion criteria:
http://dx.doi.org/10.1016/j.thromres.2015.06.031 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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(1) secondary renal disease; (2) presence of risk factor(s) for hypercoagulable state, such as the acute phase of infection, recent trauma, surgery, or cancer, and pregnancy; (3) administration of hormones, anticoagulants, or diuretics within one month; (4) treatment with plasma or albumin infusion, plasma exchange, or hemodialysis. A total of 116 MCD patients and 171MN patients received TEG examination. There were 12 MCD patients and 43 MN patients who did not meet the diagnostic criteria for NS, 31 MCD patients and 25 MN patients receiving hormones or anticoagulant therapy prior to admission, 12 MCD patients aged b18 years, and 2 MN patients with abnormal liver function who were excluded from the study. In total, 61 NS patients with MCD and 101 NS patients with MN were included, while 20 healthy adults volunteered as normal controls. The MN and MCD patients were stratified into two layers according to serum albumin levels (b20 g/l or 20–30 g/l). There were 32 MN patients and 27 MCD patients in SALB b20 g/l level, whereas 69 MN patients and 34 MCD patients in SALB 20–30 g/l level. All renal-biopsy-patients had signed the informed consent of Renal Clinical Database Establishment when hospitalized, allowing their data for clinical research. A description of the study flow is depicted in Fig. 1. 2.2. Physical and Clinical data We collected physical and clinical data from101 MN patients and 61MCD patients. These data included Body height, body mass, systolic blood pressure (SBP), diastolic blood pressure (DBP), hemoglobin (Hbg), platelet count(PLT), liver function, serum total protein, serum albumin (SALB), serum creatinine (Scr), cholesterol(CH), triglycerides(TG), serum IgG and 24-h urine protein excretion (UPr). Body mass index (BMI) was calculated in formulae as follows: BMI = weight (kg) / [height (m)]2. Mean arterial pressure (MAP) was calculated in formulae as follows: MAP = (SBP (mmHg) + DBP (mmHg) × 2) /3. The estimated glomerular filtration rate (eGFR) was calculated according to the Modification of Diet in Renal Disease (MDRD) formula: for males; eGFR ml=min=1:73 m2 −1:154 ¼ 186 ðserum creatinineðμmol =LÞ=88: 4Þ ageðyearsÞ−0:203 ; for females; eGFR ml=min=1:73 m2 −1:154 ¼ 186 ðserum creatinineðμmol=LÞ=88: 4Þ
ageðyearsÞ−0:203 0:742:
2.3. Routine coagulation parameters Activated partial thromboplastin time (APTT) and prothrombin time (PT).
(3) maximum amplitude (MA)- indicative of the strength of clot that reflects the cross interaction between platelet functions and coagulation; (4)coagulation index (CI), which represents the overall coagulation profile and is calculated from the R, K, α-angle, and MA values. The typical TEG profile for hypercoagulability is characterized by short R time and highα-angle, MA, CI [7]. 2.5. Statistical analysis Data analysis was performed using Statistical Package for Social Science (SPSS) 17 statistical software. Normally distributed continuous variables were expressed as mean ± standard deviation and nonnormally distributed variables were expressed as median (interquartile range). For continuous data, statistical analysis in patient groups and healthy control group was made by t-test, Mann–Whitney U test, Kruskal-Wallis test or ANOVA as appropriate depending on the normality and levels of the outcome variable. TEG parameters: CI, R, MA and αangle values of four patient subgroups were analyzed in factorial designed ANOVA with factors disease (two levels: MCD, MN) and SALB (two levels: b 20 g/l, 20–30 g/l). Interaction between disease and SALB was included. Categorical variables were compared using the using the χ2 test. Linear regression analysis was used to detect any relation between TEG parameters and clinical data. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Clinical features of 101 NS patients with pathological MN Data of 101 NS patients with pathological MN on age, gender, BMI, MAP, Hbg, PLT, SALB, Scr, BUN, UA, eGFR, TC, TG, UPr, IgG, C3 and Renal pathology were presented in Table 1. 3.2. Linear regression analysis between TEG parameters and clinical variables presented in Table 1 in 101 MN patients To identify the correlated factors influencing TEG parameters (R, α-angle, MA and CI) in MN patients, linear regression analysis was performed on the variables presented in Table 1. The results showed that the TEG parameters of MN patients were mainly correlated with serum albumin. As shown in Fig. 2, CI, α-angle and MA were inversely correlated with serum albumin level (r = − 0.628, P b 0.001; r = − 0.552, P b 0.001; r = − 0.369, P b 0.001). R was positively correlated with serum albumin level (r = 0.432, P b 0.001). Besides, we also found CI was positively correlated with 24-hour urine protein excretion(r = 0.378, P b 0.001) in MN patients. No significant correlation was observed between TEG parameters and age, BMI, MAP, MN stage, or levels of Hbg, PLT, CH, TG, serum IgG, or C3. 3.3. Subject Characteristics
2.4. TEG analysis Coagulation status was assessed via TEG using citrated whole-blood samples (2.0 ml sample volume per tube; 3.8% citrate in 9:1 blood to citrate ratio). For each TEG assay, citrated whole blood (1 ml) was pipetted into to a vial containing 1% kaolin, which was inverted 5 times to ensure mixing of kaolin with the blood. Then, 340 μl kaolin-activated citrated whole blood was transferred to the TEG cup to which 20 μl of 0.2 mol/l CaCl2 had been preloaded for recalcification. The TEG analyzer was stopped 40–60 minutes after reaching maximum amplitude at 37 °C. Parameters included the following: (1) reaction time (R) - time from the start of the test to a TEG amplitude of 2 mm, reflecting the combined effect of coagulation factors involved in the initiation of hemostasis; (2) α-angle- the angle between the tangent line (drawn from the split point to the curve) and the horizontal base line, representing the acceleration of fibrin build-up and cross-linking;
Study above showed that hypoalbuminemia is a risk factor for hypercoagulability in MN patients. We divided MN and MCD patients into two subclasses by the levels of SALB. Table 2 presents detailed stratified data of MN and MCD patients in different subgroups based on SALB (b20 g/l or 20–30 g/l) and clinical features of heathy volunteers. NS patients had significantly higher BMI, MAP, CH, TG and lower SALB, eGFR than healthy controls. MN and MCD patients showed no significant difference in SALB and UPr at the same SALB level. Besides,the whole study population showed no significant differences in age, gender and PLT (P N 0.05). 3.4. Routine coagulation test parameters The five groups showed no significant difference in PT value. In terms of APTT value, there was no significant difference among low
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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Fig. 1. The flow chart of this study.
level SALB MCD group (SALB b 20 g/l), high level SALB MCD group (SALB 20–30 g/l) and normal control group, but the values of these three groups are higher than that of the MN subgroups (P b 0.05). (Table 3) 3.5. TEG parameters Table 4 compares TEG parameters between control group and patient groups. R value in normal controls was significantly higher than that in MN subgroups, but was not statistically different from that in MCD subgroups. NS patients (both MCD and MN) had significantly higherα-angle, MA and CI values than healthy controls(p b 0.05). Besides, CI, R, MA and α-angle values of four NS patient subgroups (Table 4) were analyzed in factorial designed ANOVA with factors
Table 1 Clinical features of 101 NS patients with pathological MN. Clinical characteristics
Patients (n = 101)
Age (y) Gender(male/female) BMI (kg/ m2) MAP(mmHg) Hbg (g/l) PLT (109/l) SALB (g/l) Scr (umol/l) BUN (mg/dl) UA(umol/l) eGFR (ml/min/1.73 m2) TG (mmol/l) TC (mmol/l) UPr (g/day) IgG(mg/dl) C3(mg/dl) Renal pathology I I–II II III
44.94 ± 16.50 60/41 25.12 ± 4.04 96.41 ± 13.40 129.92 ± 19.22 258.25 ± 72.49 22.96 ± 3.93 67.80(57.90, 84.75) 4.79(3.65,6.18) 323.97 ± 102.59 104.64 ± 32.52 1.95(1.42, 2.75) 6.73(5.58, 8.29) 5.75 ± 2.42 474.5(323.3,672.7) 115.46 ± 27.55 55 23 19 4
Data are expressed as mean ± standard deviation (SD) or median (interquatile range) as appropriate; BMI = body mass index; MAP = Mean arterial pressure; Hbg = hemoglobin; SALB = serum albumin; CH = cholesterol; TG = triglyceride; Scr = serum creatinine; BUN = urea nitrogen; UA = uric acid; eGFR = estimated glomerular filtration rate; and UPr = 24-h urine protein excretion.
disease (two levels) and SALB (two levels). Interaction between disease (MCD, MN) and SALB (b 20 g/l, 20–30 g/l) was presented in Fig. 3. The main effect of factor disease was statistically significant (CI value: F = 32.08, P b 0.001; R value: F = 19.24, P b 0.001; MA value: F = 8.31, P = 0.005;α-angle: F = 15.15, P b 0.001) and the main effect of factor SALB was also statistically significant (CI value: F = 31.98, P b 0.001; R value: F = 14.84, P b 0.001; MA value: F = 9.28, P = 0.003;α-angle: F = 17.18, P b 0.001).But no statistically significant interaction between the two factors was found (CI value: F = 0.65, P = 0.42; R value: F = 1.30, P = 0.25; MA value: F = 0.07, P = 0.78;α-angle: F = 1.08, P = 0.30). 4. Discussion Thromboembolic events are one of the most severe complications in patients with NS. Real-time control of the coagulation status of the patient and preventions of the occurrence of thrombotic events are challenging to clinicians. Because of the complexity of NS and the limitations of coagulation test methods, the mechanisms of hypercoagulability in NS patients have not been fully elucidated. TEG serves as an approach to test coagulation that can help assess the coagulation status of the patient. To date, TEG has been clinically used for several clinical settings to evaluate and fix the coagulopathy and to study clotting mechanism. The typical TEG profile for hypercoagulability is defined by the presence of the following: shortened R time, increasedα-angle and MA. In this study, NS patients in the MCD and MN groups are characterized by shorter R times and higherα-angle, MA, CI than healthy adults, indicating that NS patients tended to be hypercoagulable, with higher severity in cases of MN. This finding is consistent with previous reports that MN is the pathological type of NS with the highest incidence thromboembolic events [2,3]. R value mainly reflects the activity of enzymatic coagulation cascade and APTT, PT represent intrinsic and extrinsic pathway respectively. This study found no difference in the R value between MCD patients and healthy controls. However, R value was significantly reduced in the MN subgroups, indicating accelerated enzymatic activity in MN patients. Combined with routine coagulation test, PT value showed no difference among MCD, MN and healthy controls whereas APTT had a consistent variation with R value, indicating hypercoagulability process does not activate extrinsic coagulation pathway but intrinsic pathway.α-angle represents velocity of clot formation
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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Fig. 2. Correlation of serum albumin with TEG parameters in the MN patients. A: Correlation of serum albumin with CI value. B: Correlation of serum albumin with R value. C: Correlation of serum albumin with α-angle value. D: Correlation of serum albumin with MA value.
and increase inα-angle may be due to fibrinogen elevation. MA mainly reflects platelet function and is influenced by the quantity and quality of platelets. The data showed all study population have no significant difference in platelet counts, whereasα-angle was significantly increased in MN and MCD patients and the MA value increased in the order of MN N MCD N healthy control group. The results indicate that increased fibrinogen, abnormal platelet activation and accelerated fibrin-platelet
interaction in MN and MCD patients, especially the former group. Thus, our findings suggest that the hypercoagulable state in MCD patients may be that the coagulation factors preceding the initial fibrin platelet interaction were not fully activated, whereas the hypercoagulable state in MN patients involves the whole thrombotic processes acceleration (accelerated intrinsic pathway, abnormal platelet activation and accelerated fibrin-platelet interaction). To date, studies on the roles of
Table 2 Clinical parameters of MN, MCD patients in different SALB levels and healthy controlsAbbreviations as in Table 1. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
N Age(year) Gender(M/F) BMI(kg/m2) MAP(mmHg) Hbg(g/L) PLT(10^9/L) SALB (g/L) TG(mmol/l) CH(mmol/l) Scr(umol/l) eGFR (ml/min/1.73 m2) UPr(g/day)
20 37.3 ± 13.8 11/9 21.6 ± 2.9 86.9 ± 7.6 137.1 ± 14.0 253.3 ± 53.7 44.4 ± 2.8 1.1 ± 0.5 3.8 ± 0.8 62.7 ± 8.6 121.4 ± 12.8
27 41.2 ± 16.5 16/11 25.2 ± 3.2★ 93.8 ± 10.4★ 135.6 ± 18.2◆ 264.1 ± 57.1 17.1 ± 1.9★# 2.2(1.7, 2.9)★◆ 9.9(7.6, 11.4)★ 79.5(60.7, 96.4)★ 89.3 ± 28.1★#
32 43.6 ± 18.7 19/13 25.6 ± 4.8★ 98.2 ± 13.7★ 125.1 ± 17.3★ 273.7 ± 64.7 18.2 ± 1.9★※ 2.0(1.6, 2.5)★ 7.6(5.8, 9.3)★ 69.6(60.1, 85.3)★ 99.7 ± 33.6★
34 35.9 ± 13.1 21/13 25.2 ± 4.1★ 92.1 ± 14.2★ 138.1 ± 17.1 248.8 ± 69.0 24.1 ± 2.8★ 2.3(1.8, 3.3)★▲ 8.7(7.1, 10.1)★ 67.9(52.8, 85.6)★ 111.1 ± 36.8★
69 45.5 ± 15.3 41/28 24.9 ± 4.5★ 95.3 ± 13.2★ 132.3 ± 19.6 250.1 ± 75.0 25.2 ± 2.3★ 1.9(1.3, 2.9)★ 6.6(5.3, 8.0)★ 65.0(57.1, 84.7)★ 106.5 ± 31.8★
---
6.3(4.7, 8.4)
7.4(6.2, 8.9)※
5.7(3.6,7.7)
4.3(3.7, 5.34)
★ ◆ ▲ ※
#
P b 0.05, vs control group; P b 0.05, MCD with SALB b20 g/l group vs MN with SALB b20 g/l group; P b 0.05, MCD with SALB 20–30 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MN with SALB b20 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MCD with SALB b20 g/l group vs MCD with SALB 20–30 g/l group.
Table 3 Comparison of Routine coagulation test parameters in study population. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
APTT(s) PT(s)
39.7 ± 2.0 12.8 ± 0.40
39.6 ± 3.3◆ 12.8 ± 0.7
34.8 ± 4.9★ 12.7 ± 0.9
37.0 ± 5.4▲ 12.4 ± 0.7
34.7 ± 7.5★ 12.3 ± 0.6
※
P b 0.05, MN with SALB b20 g/l group vs MN with SALB 20–30 g/l group; P b 0.05, MCD with SALB b20 g/l group vs MCD with SALB 20–30 g/l group. ★ P b 0.05, vs control group; ◆ P b 0.05, MCD with SALB b20 g/l group vs MN with SALB b20 g/l group; ▲ P b 0.05, MCD with SALB 20–30 g/l group vs MN with SALB 20–30 g/l group;
#
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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Table 4 Comparison of TEG parameters between control group and patient groups. Variables
Controls
MCD patients with SALB b20 g/l
MN patients with SALB b20 g/l
MCD patients with SALB 20–30 g/l
MN patients with SALB 20–30 g/l
R (min) α-angle (deg) MA (mm) CI
7.3 ± 1.1 57.7 ± 5.6 56.6 ± 4.9 −1.9 ± 1.7
6.7 ± 1.0 68.9 ± 5.1★ 71.1 ± 5.4★ 1.24 ± 1.3★
5.5 ± 0.8★ 74.8 ± 3.2★ 73.3 ± 4.8★ 2.8 ± 1.1★
7.2 ± 1.5 65.5 ± 6.6★ 68.1 ± 5.0★ 0.1 ± 1.98★
6.6 ± 1.2★ 68.9 ± 6.2★ 70.9 ± 5.4★ 1.24 ± 1.2★
★
P b 0.05, vs control group.
platelets, fibrinogen and coagulation factors in the hypercoagulability of NS patients have been conducted independently [8–15], limiting our understanding on the comprehensive assessment of coagulation. The present study was the first to use TEG analysis to investigate the effects of global hemostasis assay on the mechanisms of hypercoagulability in patients with different pathological types of NS. The linear regression analysis for hypercoagulability in MN patients showed that hypoalbuminemia was the major risk factor for thrombophilia of the nephrotic syndrome, and further TEG parameters of four NS patient subgroups analyzed in factorial designed ANOVA confirmed that hypercoagulable state of MN patients was accompanied by a decrease in serum albumin. This result confirms previous findings in the literature [15] and suggests a need to strengthen efforts to prevent hypercoagulable state, especially for MN patients with severe hypoalbuminemia. Because of the relatively low correlation coefficient between CI and 24-hour urinary protein excretion, we could not clearly demonstrate urine protein as a risk factor for hypercoagulability [16]. Our study has some potential limitations. Due to its retrospective study design, samples may not well present patient population. We
enrolled MN and MCD patients who had taken TEG examination. As patients who had high clinical suspicion of hypercoagulability might easily undergo TEG examination, our findings may overestimate the degree of hypercoagulable state. Besides, some information for potential hypercoagulability risk factors such as medication history of hormonal contraceptives was not able to obtain. The result of our study is the assessment of potential risk for hypercoagulability by TEG in NS patients with MN. We did not follow up and assess the clinical incidence of thrombosis in the observed patients. Thus the study is not sufficient to use TEG to resolve potential risk for thrombosis in MN patients. This study documents that patients with MN tend to be more hypercoagulable than normal or patients with MCD. The hypercoagulable state in MN patients involves the whole thrombotic processes acceleration (activated intrinsic pathway,increased fibrinogen, abnormal platelet activation and accelerated fibrin-platelet interaction), whereas hypercoagulable state in MCD patients may be that the coagulation factors preceding the initial fibrin platelet interaction are not fully activated. Thus, this study based on TEG analysis has implications for guiding clinical interventions for hypercoagulability in NS. Concomitant
Fig. 3. Main effect and interaction between two factors: disease and SALB. A: Effect of disease and SALB on CI value. B: Effect of disease and SALB on R value. C: Effect of disease and SALB on MA value. D: Effect of disease and SALB on Angle value.
Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
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anticoagulant and antiplatelet therapy is required for MN patients, while antiplatelet therapy should be emphasized for MCD patients. Since hypercoagulable state is accompanied by a decrease in serum albumin, greater efforts should be made for MN patients with severe hypoalbuminemia.
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Please cite this article as: M. Huang, et al., Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.031
