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ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 2  |  Page : 689-694

Study of levels of protein Z as a risk factor for thrombosis in children with nephrotic syndrome


1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt

Date of Submission01-Nov-2016
Date of Acceptance16-Feb-2017
Date of Web Publication27-Aug-2018

Correspondence Address:
Mohammed H Youssef
33 Abd El-Rehem Abd El-Hai St., Shebin El-Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_589_16

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  Abstract 


Objective
The aim of this study is to assess the level of protein Z (PZ) as a risk factor for thrombosis in children with nephrotic syndrome (NS).
Background
NS results from any of several well-described primary glomerulopathies that are defined by histopathology and clinical criteria. The main pathomechanism of complications originates from the large loss of plasma proteins in the urine of nephrotic children, and thromboembolism is among the most serious complications. PZ plays an important role in inhibiting coagulation, serving as a cofactor for the inactivation of activated factor X by plasma-PZ-dependent protease inhibitor. Decreased PZ may contribute to an increased risk of thromboembolic complications in children with NS.
Patient and methods
The study included 45 children divided into three groups: group I included 15 patients in active stage of NS, group II included 15 patients in remission stage, and group III included 15 apparently healthy age-matched and sex-matched patients as controls. Patients were subjected to the following: history taking and clinical examination, assessment of weight, height, and edema, platelet count, prothrombin time, activated partial thromboplastin time, creatinine, urine protein/creatinine ratio, serum albumin, cholesterol, and antithrombin III (ATIII) level assay by radial immunodiffusion and PZ level assay by enzyme-linked immunosorbent assay.
Results
There were highly statistical significant differences regarding platelet count (P = 0.000), urine protein/creatinine ratio (P = 0.000), serum albumin (P = 0.000), cholesterol (P = 0.000), ATIII (P = 0.000), and PZ (P = 0.000) between the studied groups.
Conclusion
PZ level in plasma and ATIII level were found to be lower in children with NS in active stage, and this may contribute to an increased risk of thromboembolic complications in children with NS.

Keywords: nephrotic syndrome, protein Z, thromboembolic complications


How to cite this article:
Khatab AA, El-Mashad GM, Khodair SZ, Youssef MH. Study of levels of protein Z as a risk factor for thrombosis in children with nephrotic syndrome. Menoufia Med J 2018;31:689-94

How to cite this URL:
Khatab AA, El-Mashad GM, Khodair SZ, Youssef MH. Study of levels of protein Z as a risk factor for thrombosis in children with nephrotic syndrome. Menoufia Med J [serial online] 2018 [cited 2018 Nov 19];31:689-94. Available from: http://www.mmj.eg.net/text.asp?2018/31/2/689/239746




  Introduction Top


Nephrotic syndrome (NS) is primarily a pediatric disorder and is 15 times more common in children than in adults. The characteristic features of NS are heavy proteinuria, hypoalbuminemia, edema, and hyperlipidemia [1]. Complications may arise as a result of the disease itself or secondary to treatment. There are five major complications directly related to the nephrotic state, including severe infections, thromboembolism, renal impairment, anasarca, hypovolemia, and growth retardation [2].

Thromboembolic disease is an important complication in both children and adults, which is seen in 10% of adults and in ∼5% of children with NS. Although these complications are less common in children, the consequences are more severe. They have been attributed to an imbalance between procoagulant/thrombotic, anticoagulant/antithrombotic factors, thrombocytosis, hemoconcentration, hyperviscosity, relative immobilization, and therapy with corticosteroids and diuretics [3].

Protein Z (PZ) is a vitamin-K-dependent, calcium-ion (Ca ++)-binding protein that is synthesized by the liver [4]. The physiological function of PZ was uncertain for many years until in-vitro and in-vivo studies demonstrated that PZ has an important role in inhibiting coagulation, serving as a cofactor for the inactivation of activated factor X by plasma-PZ-dependent protease inhibitor [5]. Several authors have shown that low PZ level is a risk factor for thrombosis, although not all studies confirmed this [3].

The aim of this study was to assess levels of PZ as a risk factor for thrombosis in children with NS.


  Patients and Methods Top


The present study was carried out on 45 children: 30 children with NS who attended the Nephrology Unit of Pediatric Department of Menoufia University Hospital from April 2015 to April 2016 and 15 apparently healthy children as controls. Their ages ranged from 2 to 10 years. Ethics rules in the form of approval of the ethics committee of Menoufia University Hospitals and patient consent were applied on the studied groups. They were divided into three groups:

  1. Group I: this group included 15 patients in the active stage of disease according to criteria of activity [6]; nine were boys, whereas six were girls, and their ages ranged from 2.5 to 9 years
  2. Group II: this group included 15 patients in remission; 10 were boys, whereas five were girls, and their ages ranged from 2 to 10 years
  3. Group III: this group included 15 apparently healthy children with matched age and sex as a control group.


All participants in this study were subjected to complete history taking laying stress on symptoms of NS (puffiness in eye lids, lower limb swelling, and ascites). Thorough clinical examination was performed stressing on signs of NS (lower limb edema, ascites, and scrotal edema) and manifestations of thromboembolic disease by examination of peripheral pulsations, color change, and examination of venous system to exclude venous thrombosis or varicose vein. Hematological and biochemical investigations included total platelet count, prothrombin time (PT) and activated partial thromboplastin time (aPTT), serum creatinine, 24-h urine collection for protein excretion, serum albumin level and serum cholesterol level, antithrombin III (ATIII) level by radial immunodiffusion assay, and PZ levels by enzyme-linked immunosorbent assay (ELISA).

Sampling

Five milliliter of blood sample was collected from each individual under complete aseptic condition by clean venipuncture. The sample was divided as follows: 2.5 ml of whole blood was added to a plain sterile tube and allowed to clot at 37°C. The serum was separated by centrifugation and used for estimation of creatinine, albumin, cholesterol, and ATIII by immunodiffusion and 2.5 ml of whole blood was added to a citrate-contained sterile tube. Plasma was separated by centrifugation and used for estimation of PT, aPTT, and assay of PZ level by ELISA.

Laboratory methods

Biochemical tests for detection of serum creatinine, albumin, and cholesterol were performed using a Synchron CX9 Autoanalyzer (Beckman Coulter Inc., Brea, California, USA). Meanwhile, total platelet count was analyzed by Pentra XL80 Analyzer (ABX Diagnostics, Montpellier, France). aPTT and PT (STAGO, Asnières sur Seine Cedex, France) were performed, and 24-h urine collection for protein excretion was performed for assessment of protein/creatinine ratio. PZ levels in plasma were detected by ELISA. ATIII level in serum was detected by radial immunodiffusion assay.

Protein Z estimation by enzyme-linked immunosorbent assay

The Assay Max Human PZ ELISA kit (EP3333-1; Assaypro LLC, Missouri, USA) is designed for detection of human PZ in plasma, serum, urine, milk, and cell culture samples. This assay uses a quantitative sandwich enzyme immunoassay technique that measures human PZ in less than 4 h. A polyclonal antibody specific for human PZ has been precoated onto a 96-well microplate with removable strips. PZ in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for PZ, which is recognized by a streptavidin–peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Antithrombin III level estimation by radial immunodiffusion assay using DIFFU-PLATE

The procedure consists in an immunoprecipitation in agarose between an antigen and its homologous antibody. It is performed by incorporating one of the two immunoreactants (usually antibody) uniformly throughout a layer of agarose gel, and then introducing the other reactants (usually antigen) into wells duly punched in the gel. Antigen diffuses radially out of the well into the surrounding gel–antibody mixture, and a visible ring of precipitation forms where the antigen and antibody reacted (ND11-12; Biocientifica S.A., Buenos Aires, Argentina).

Statistical analysis

The collected data were analyzed using SPSS software version 20 (IBM SPSS Statistics for Mac, Released 2011; IBM Corp., Armonk, New York, USA). Descriptive statistics in the form of mean ± SD was used for parametric data. c2-Test was used for qualitative variables. Analysis of variance test was performed for comparison between three or more groups having normally distributed quantitative variables, and Kruskal–Wallis test was used for comparison between three or more groups not normally distributed having quantitative variables. Spearman's correlation coefficient (r) (nonparametric test) is a test used to measure the association between two quantitative variables. Confidence interval (CI) was calculated by logistic regression analysis. The significance level was set at 0.05 or less. The receiver operating characteristic (ROC) curve is a graphic representation of the relationship between sensitivity and specificity at different cutoff points for diagnostic test. It was used in this study to measure the cutoff point at which PZ level predicts active NS.


  Results Top


In the present study, there was no statistically significant difference between the studied groups regarding age and sex (P = 0.6 and 0.91, respectively). There was no statistically significant difference regarding weight and height between the studied groups (P = 0.18 and 0.64, respectively). It also shows that there was a highly statistically significant difference regarding edema between the studied groups (P = 0.000), with no difference regarding hypertension (P = 0.12). None of the patients had signs of thrombosis [Table 1].
Table 1: Clinical examination of the studied groups

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In [Table 2], there were highly statistically significant differences regarding platelet count between the studied groups, where the platelet count in children with NS in active stage was markedly higher than patients in remission (P = 0.000) and healthy controls (P = 0.000). There were also highly statistically significant differences regarding urine protein/urine creatinine ratio between the studied groups, where the urine protein/creatinine ratio in children with NS in active stage was markedly higher than patients in remission (P = 0.000) and healthy controls (P = 0.000). There were also highly statistically significant differences regarding serum albumin between the studied groups, where the serum albumin in children with NS in active stage was markedly lower than patients in remission (P = 0.000) and healthy controls (P = 0.000). In addition, there were also highly statistically significant differences regarding serum cholesterol level between the studied groups, where the serum cholesterol level in children with NS in the active stage was markedly higher than patients in remission (P = 0.000) and healthy controls (P = 0.000). There were no statistically significant differences as regards PT, aPTT, and serum creatinine between the studied groups.
Table 2: Hematological and biochemical tests of the studied groups

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There was a highly statistically significant difference regarding PZ level between the studied groups, where the plasma PZ level in children with NS in active stage was markedly lower than patients in remission (P = 0.000) and healthy controls (P = 0.000) [Table 3].
Table 3: Comparison between the studied groups regarding levels of protein Z and antithrombin III

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There were highly statistically significant differences regarding ATIII level between the studied groups, where ATIII level in children with NS in active stage was markedly lower than patients in remission (P = 0.000) and healthy controls (P = 0.000) [Table 3].

There was no significant correlation between PZ in plasma and other variables in the active nephrotic cases. ATIII is statistically highly negatively correlated with aPTT in the active nephrotic cases. In addition, it shows a statistically negative correlation with protein/creatinine ratio. In addition, it shows no significant correlation with the other variables.

The optimal cutoff point of PZ level in active NS was obtained by ROC curve: 2.05 with 93% sensitivity, 77% specificity, and 82% accuracy with CI 0.84–1.00 [Table 4] and [Figure 1].
Table 4: Diagnostic validity of protein Z in active nephrotic cases

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Figure 1: Receiver operating characteristic (ROC) curve for the detection of the best cutoff point of serum protein Z in active nephrotic cases.

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  Discussion Top


In the present study, regarding biochemical tests, there was no significant difference in creatinine levels between studied groups. There was a highly significant difference in albumin levels between studied groups (P = 0.000), where the level of albumin in children with NS in active stage (1.55 ± 0.29 g/dl) was markedly lower than patients in remission (3.85 ± 0.44 g/dl; P = 0.000) and healthy controls (4.35 ± 0.44 g/dl; P = 0.000). In addition, there was a highly significant difference in cholesterol levels between studied groups (P = 0.000), where the level of cholesterol in children with NS in active stage (565.27 ± 62.27 mg/dl) was markedly higher than patients in remission (172.53 ± 24.4 mg/dl; P = 0.000) and healthy controls (141.13 ± 22.18 mg/dl; P = 0.000). These results agreed with those of Gulleroglu et al. [7] who showed the same results.

In present study, other hemostatic parameters such as PT and aPTT in children with NS in active stage were not significantly different from those of patients in remission or healthy controls. This agrees with the studies by Yalçinkaya et al. [8], Al-Mugeiren et al. [9], and Özkaya et al. [3], who observed the same result, but this result does not agree with those of Ueda et al. [10], Anand et al. [11], and Mittal et al. [12], who observed that aPTT is prolonged in patients with NS in active stage than patients in remission or healthy controls; however, they observed that PT in active patients is not different from that of patients in remission or healthy controls. This may be because of the small sample size.

However, the platelet count was highly significantly different between the studied groups (P = 0.000), where the platelet count in children with NS in active stage was markedly higher than patients in remission (P = 0.000) and healthy controls (P = 0.000). This result does not agree with those of Özkaya et al. [3], who observed that the platelet count was not significantly different between the studied groups (P = 0.09). This may be because of the different age range between both groups. However, this result agrees with that of Gulleroglu et al. [7], who observed a significant difference between the studied groups (P = 0.02).

In the present study, plasma level of PZ in children with NS in active stage (1.67 ± 0.12 μg/ml) was markedly lower than patients in remission (2.68 ± 0.33 μg/ml; P = 0.000) and healthy controls (3.08 ± 0.65 μg/ml; P = 0.000). This result agrees with that of Özkaya et al. [3], who observed that the plasma level of PZ in active nephrotic patient was significantly lower than in patients in remission and healthy controls (P = 0.014 and 0.03, respectively). This result can be explained by the low molecular weight of PZ (62 kDa), and thus it can be lost in urine during the activity of NS. Therefore, the plasma level in active patients is markedly lower than remission patients and healthy controls.

In the present study, the plasma level of ATIII in children with NS in active stage (group I) (43.8 ± 20.07%) was markedly lower than patients in remission (74.73 ± 13.69%; P = 0.000) and healthy controls (87.53 ± 8.14%; P = 0.000). This result agrees with that of Özkaya et al. [3], who observed that plasma level of ATIII in active nephrotic patient is significantly lower than patients in remission and healthy controls (P = 0.009 and 0.005, respectively). These results also agree with those of Anand et al. [11], Citak et al. [13], Wygledowska et al. [14], and Prandota et al. [15].

In the present study, PZ in plasma of active nephrotic patients (group I) was not significantly correlated with serum albumin, urine protein/creatinine ratio, and ATIII. This is in contrast to the study by Özkaya et al. [3], who observed a positive correlation between PZ in plasma and serum albumin and ATIII (r = 0.53; P = 0.003) (r = 0.037; P = 0.04) and a significant negative correlation between PZ in plasma and urine protein/creatinine ratio (r=−0.51; P = 0.004) in this group. This may be because of the short duration of the study and small sample size.

In the present study, ATIII in active nephrotic patients (group I) was significantly negative correlated with urine protein/creatinine ratio (r=−0.57; P = 0.02). This result agrees with that of Özkaya et al. [3], who observed a negative correlation between ATIII and urine protein/creatinine ratio (r=−0.51; P = 0.003).

On the other hand, in this study, ATIII in active nephrotic patients (group I) was not significantly correlated with serum albumin. This is in contrast to the studies by Ozkaya et al. [3] and Yalçinkaya et al. [8], who observed a positive correlation between ATIII and serum albumin (r = 0.50; P = 0.0039 and r = 0.807; P = 0.000, respectively). This may be because of differences in races between the studies.

In the present study, diagnostic validity of PZ in diagnosis of active nephrotic cases was estimated using ROC curve for detection of the best cutoff point of PZ for diagnosis of active NS, which was 2.05 with 93% sensitivity, 77% specificity, and 82% accuracy with CI 0.84–1.00.


  Conclusion Top


In view of this study, it was concluded that PZ level in plasma and ATIII level were found to be lower in children with NS in active stage, and this may contribute to an increased risk of thromboembolic complications in children with NS.

Acknowledgements

Professor Dr Ahmed A. Khatab contributed to concepts, design, definition of intellectual content, data analysis, statistical analysis, manuscript review, manuscript editing, guarantor. Assistant Professor Dr Ghada M. El-Mashad contributed to concepts, design, definition of intellectual content, clinical studies, experimental studies, data acquisition, data analysis, statistical analysis, manuscript preparation, manuscript review, manuscript editing, guarantor. Assistant Professor Dr Seham Z. Khodair contributed to concepts, design, definition of intellectual content, data acquisition, manuscript review. Mohammed H. Youssef contributed to design, definition of intellectual content, literature search, clinical studies, experimental studies, data acquisition, data analysis, statistical analysis, manuscript preparation, manuscript editing, guarantor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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