Nephortic syndrome is a primarily pediatric disorder with an incidence of 2-3/100 children per year. The characteristic features of nephrotic syndrome are heavy proteinuria (> 40 mg m-2 h-1), hypoalbuminemia (<2.5 g dL-1), edema and hyperlipidemia (Vogt and Avner, 2004).
A hypercoagulable state and the risk of thromboembolism in both arterial and venous circulation are relatively frequent and serious features of nephrotic syndrome in children (Citak et al., 2000; Gangakhedkar et al., 2005).
Recently Tissue Factor (TF) and its inhibitor (TFPI) had been reported to participate in the process of coagulation in various diseases such as angina pectoris, end stage renal disease and insulin dependent diabetes mellitus (Prandoni, 2005).
Proinflammatory effects of TF, which appear to be independent of its procoagulant function, have been demonstrated in crescentic glomerulonephritis. Moreover, the binding of factor VIIa (FVIIa) to TF may be an important intracellular signaling mechanism that increases reactive oxygen species production by macrophages (Cunningham et al., 1995).
Recombinant TFPI has been shown to attenuate the lethal inflammatory and coagulation responses (Al-Mugeiren et al., 2006). Several studies have demonstrated high serum concentration of TF and TFPI in nephrotic patients. They suggested that, the increase in TFPI might attenuate but not abolish the action of TF on the vascular endothelium (Malyszko et al., 1999).
Heparin is known to exert its antithrombotic effect by accelerating the effect of antithrombin (AT) and by mobilizing TFPI into the circulation from vascular endothelium. Recently, heparin has been reported to decrease FVIIa, this was suggested as a new antithrombotic action of heparin and that FVIIa assay is more sensitive than TFPI and AT assays during heparin treatment (Hansen et al., 2000).
The aim of this study was to evaluate the degree of the hypercoagulation in children with nephrotic syndrome by measuring serum TF and FVIIa and to study the role of heparin in inhibiting the coagulation action of TF either directly or indirectly via inhibition of FVIIa and its effect on the clinical condition of the patients.
MATERIALS AND METHODS
This follow up study comprised 40 children with primary nephrotic syndrome and 15 controls. They were classified into two groups.
Patients (group I): Included 40 nephrotic children all were in activity at the start of the study. Group I was further subdivided according to the treatment protocol into two groups:
Group IA: This group received low molecular weight heparin (LMWH) (50 unit kg-1 body weight single daily dose subcutaneously for six days) beside steroid therapy as prednisone 60 mg m-2 orally daily. This group included 20 nephrotic patients (13 males and 7 females); their ages ranged form 3.5-15.0 years (mean±SD = 9.0±3.9). Twelve of them were steroid dependent and eight were steroid resistant. Duration of illness ranged form 2-120 months (mean±SD = 45.7±40.2).
Group IB: This group received steroid therapy only. It included 20 nephrotic patients (12 males and 8 females); their ages ranged form 2.5-12.0 years (mean±SD = 7.8±3.2). Twelve of them were steroid dependent and eight were steroid resistant. Duration of illness ranged form 5-108 months (mean±SD = 41.6±36.0).
Exclusion criteria for the patients: Patients with abnormal kidney function (creatinine clearance <80 mL-1 min), secondary nephrotic syndrome, chronic liver disease, prolonged PT or PTT, platelet dysfunction, history of active bleeding or thrombosis, severe hypertension and recipients of cytotoxic drugs were excluded from the study.
Controls (group II): It included 15 apparently healthy age and sex matched children. They were 9 males and 6 females. Their ages ranged from 4.0-11.0 years (mean±SD = 8.3±2.4).
All patients were evaluated as follows
||Full history taking.
||Thorough clinical examination.
||Laboratory investigations: Complete urine analysis, 24 h urinary proteins,
Complete blood count, ESR, Total serum protein and serum albumin, serum
cholesterol, serum creatinine and creatinine clearance, blood urea nitrogen,
Prothrombin Time (PT) and partial thromboplastin time (PTT), serum Tissue
Factor (TF) and serum FVIIa by ELISA.
The clinical assessment and laboratory investigations were done at day 1, 7 and 30.
Statistical methods: Descriptive and analytical statistics were performed
on IBM-compatible computer by using SPSS 11.5 software package. Continuous data
were presented as mean±standard deviation (mean±SD), median and
range. For continuous non-parametric data, Mann-Whitney (U) test was used for
inter-group analysis and Spearman correlation coefficient (r) tests were used
for intra-group analysis. Inter-group comparisons of categorical data were performed
by using chi square test (χ2 value). p-values < 0.05 were
considered significant and p value <0.01 and <0.001 were considered highly
Table (1 and 2) and Fig.
(1 and 2) show the levels of serum TF and FVIIa among
the different studied groups. At day 1, both serum TF and FVIIa levels showed
no significant difference between GIA and GIB patients (p>0.05). Both groups
showed significantly higher serum TF and FVIIa levels as compared to the control
group (p<0.01). At day 7, GIA had significant decrease in serum TF and FVIIa
levels as compared to GIB (p<0.01 and p<0.05, respectively). However,
serum TF and FVIIa in GIA was still higher than that of the control group (p<0.01).
||Serum tissue factor in the studied groups during the follow
|* = Non significant ** = Highly significant
|| Serum FVIIa in the studied groups during the follow up period
|* = Significant, ** = Highly significant
Further decrease in serum TF and FVIIa levels in GIA as compared to GIB patients
(p<0.01) was observed at day 30 and was comparable with the values of the
control group (p>0.05). Meanwhile, in GIB patients, the values of serum TF
at day 30 were still significantly higher than the control group (p>0.01).
At the start of the study, serum TF and FVIIa levels were comparable between
steroid resistant and steroid dependent nephrotic patients (p>0.05 in each
comparison). At day 1, there was no significant difference in serum TF and FVIIa
results between group IA and group IB in both steroid dependant and steroid
resistant patients (p>0.05 in each comparison). At day 1 and 7, steroid dependent
and steroid resistant nephrotic patients of groups IA and IB showed significantly
higher serum TF and FVIIa levels as compared to the control group (p<0.01)
in each comparison except in comparison of serum FVIIa in group IA steroid resistant
patients at day 7 versus control group, p<0.05). At day 30, steroid dependent
and steroid resistant nephrotic patients of group IA had comparable serum TF
and FVIIa levels with the control group (p>0.05 in each comparison).
||Serum tissue factor in both patients groups during follow
up period [*= highly significant increase versus control group (p<0.01)]
||Serum factor VIIa in both patients groups during follow
up period [*= highly significant increase versus control group (p<0.01)]
On the contrary, steroid dependent and steroid resistant nephrotic patients
of group IB had still significantly higher levels of both serum TF and FVIIa
as compared to the control group (p<0.01 in each comparison).
At the start of the study, serum albumin results were comparable in both group IA and group IB patients (p>0.05). In addition, significant negative correlation was found between serum albumin and both serum TF and serum FVIIa in all nephrotic patients at the start of the study (p<0.05 and p<0.001, respectively). At day 7 and 30, serum albumin was significantly higher in group IA as compared to group IB (p<0.01 in each comparison).
As regards 24 h urinary proteins results at day 1, there was no significant difference between both groups (p>0.05). At day 7, 30, 24 h urinary proteins were significantly lower in group IA as compared to group IB (p<0.01 in each comparison). At day 1,7 and 30, there was no significant difference between both patients groups in the serum cholesterol levels (p>0.05). However, significant positive correlation was found between serum cholesterol level and both serum TF and FVIIa levels in all nephrotic patients at the start of the study (p<0.05 in each correlation).
At the end of the study period (30 days), 35 of our patients were in clinical remission as evidenced by disappearance of both edema and proteinuria. Group IA patients showed significantly shorter time to remission compared with group IB (p<0.05). Time to remission was significantly reduced in steroid resistant patients who received LMWH as compared to steroid resistant patients who received steroids only (p<0.05).
The proposed role of TF and FVIIa in the pathogenesis of glomerular diseases has urged us to evaluate their levels in the serum nephrotic children and to investigate the possible alteration in their levels in relation to heparin treatment and disease activity.
Our results revealed that, in all nephrotic patients before starting heparin therapy, serum levels of TF and FVIIa were significantly higher as compared to the control group (p<0.01). This finding denotes increased activity of the coagulation pathway during the stage of active nephrosis. The elevated levels of serum TF and FVIIa in our series are in accordance with those observed by Malyszko et al. (1999) on nephrotic syndrome.
On the first day of the current study, serum TF levels and FVIIa were positively correlated with the values of 24 h urinary proteins and were negatively correlated with serum albumin levels in all nephrotic children. This finding raises in mind the well established relationship between the severity of albuminuria and progressive renal disease (Assadi, 2005; de Jong and Brenner, 2004) and the possible link between elevated serum TF level and FVIIa and the severity of glomerular injury.
A positive correlation between cholesterol and both TF and FVIIa was observed in all nephrotic patients at the start of our study. This is in accordance with other studies (Puccetti et al., 2000; Camera et al., 2002) that concluded that, the prothrombotic phenotype of arterial wall associated with elevated serum cholesterol levels, is mediated by TF over expression and they demonstrated that, prothrombotic tendency was reduced through inhibition of TF synthesis. Moreover, Jeanpierre et al. (2003) analyzed the effect of dietary lipid lowering on TF expression. They showed that, dietary lipid lowering decreases the thrombotic potential of ruptured atherosclerotic plaques through TF decrease. In addition, Chi et al. (2004) concluded that, thrombus formation is initiated by the activation of TF/FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVIIa coagulant activity.
Follow up of the clinical parameters and the laboratory results after 7 days from the start of the study revealed obvious clinical improvement in group IA as compared to group IB in the form of significant weight reduction, fading of edema, significant increase in serum albumin and decrease in urinary proteins. This was also accompanied by significant decrease in serum TF (p<0.01) and FVIIa (p<0.05). Therefore, LMWH therapy induced a more rapid improvement in both clinical and laboratory markers of remission and this improvement was parallel with the reduction of serum TF and FVIIa.
Moreover, the laboratory improvement was more marked after 30 days of follow up since serum albumin was significantly higher and the 24 h urinary proteins were significantly lower in group IA as compared to group IB. Furthermore, significant reduction in serum TF and FVIIa was observed in group IA as compared to group IB (p<0.01) to the extent that serum TF and FVIIa levels reached the control baseline (p>0.05). Therefore, the beneficial effects of LMWH extended beyond the period of its administration since the improvement in the laboratory parameters was maintained until the end of the study.
Steroid resistant nephrotic syndrome (SRNS) causes morbidity and mortality due to persistent edema, hypertension and progression to renal failure (Crew et al., 2004).
In the present study, the beneficial effect of LMWH in SRNS was evident. Steroid
resistant patients of group IA had serum TF and FVIIa levels comparable with
control values at day 30 of the study. Meanwhile, the corresponding values of
steroid resistant patients in group IB remained significantly higher than the
controls at that time. Also, a significant reduction in time to induce remission
among patients with SRNS who received heparin was observed. This finding highlights
the value of heparin therapy in SRNS and supports the finding of Mori et
al. (2004) who demonstrated in their study that methyl prednisolone therapy
with heparin can induce remission in children with SRNS, even when the patient
is resistant to cyclophosphamide or cyclosporine A.
Several studies have been conducted on adults demonstrating the efficiency of heparin as a useful agent in reducing the duration of proteinuria and edema (Crew et al., 2004; Appel, 2006) based on the fact that, heparins exert an anti-inflammatory action through stopping adhesion of leucocytes to endothelial surfaces, they are anticomplementary, they modulate the activities of phagocytes and they stop the proliferation of mesangial cells on vascular smooth muscle cells. Heparin prevents the release of endothelin-1 and potentiates the action of constitutive nitric oxide (Dos Santos et al., 2006).
Although none of the children in our study had evidence of thromboembolism, yet they showed significant elevation of serum TF and FVIIa levels, such disturbance predispose them to the risk of thrombotic complications.
In conclusion, therapy with LMWH led to decrease in serum TF and FVIIa. The beneficial effects of LMWH extended beyond the period of its administration since the improvement in the laboratory parameters was maintained until the end of the study. It produced significant reduction in proteinuria and elevation of serum albumin. Moreover, LMWH led to significant clinical improvement in the course of nephrotic syndrome in the form of shorter time to induce remission, thus having steroid sparing effect without bleeding complications or rebound thrombosis.