Atherosclerosis 233 (2014) 704e706
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Endothelial dysfuntion in children with idiopathic nephrotic syndrome Bharat Sharma a, Abhijeet Saha a, *, N.K. Dubey a, Kanika Kapoor a, Anubhuti b, Vinita Vijay Batra c, Ashish Datt Upadhayay d a
Division of Pediatric Nephrology, Department of Pediatrics, Postgraduate Institute of Medical Education & Research, Dr Ram Manohar Lohia Hospital, New Delhi, India Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Dr Ram Manohar Lohia Hospital, New Delhi, India c Department of Pathology, GB Pant Hospital, New Delhi, India d Department of Biostatistics, AIIMS, New Delhi, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2013 Received in revised form 21 January 2014 Accepted 30 January 2014 Available online 11 February 2014
Background: Impaired endothelial function is the initial step in atherogenesis, which is largely responsible for ischaemic heart disease and thrombotic strokes decades later. Methods: Fourty two children with first episode nephrotic syndrome (FENS) aged 1e16 years and 40 controls were enrolled. Soluble thrombomodulin (sTM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and von-willebrand factor (vWF) levels were measured in plasma in FENS, at 12 weeks of drug induced remission and in steroid resistant nephrotic syndrome (SRNS) patients at diagnosis. Results: PAI-1, sTM, vWF and t-PA were significantly raised at the onset of nephrotic syndrome (p < 0.0001). All the markers had a fall after 12 weeks of steroid treatment, but were still raised. Children with SRNS had higher levels of sTM, tPA, vWF as compared to infrequent relapsers, at onset and at 4 weeks of steroid treatment. Conclusion: Children with idiopathic nephrotic syndrome have endothelial dysfunction which is largely dependent upon disease activity. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endothelial dysfunction Nephrotic syndrome Children
1. Introduction Long term studies in adult nephrotic population indicate association of cardiovascular diseases in these patients [1]. Ordonez et al has confirmed that patients with nephrotic syndrome have a 5.5 fold increased relative risk of cardiovascular infarction, resulting in an almost threefold increase in cardiac mortality [2]. Risk factors for early atherosclerosis in adult population include hyperhomocystenemia, proteins of chronic inflammatory process, prothrombotic factors, and adhesion molecules [3]. Many of these risk factors appear in paediatric patients at the time of first occurrence or during aggravation of idiopathic nephrotic syndrome (INS) [4]. Their appearance is related to excessive loss of albumin in urine, oxidative stress, or to steroid therapy (leading to obesity, glucose utilisation disorders, and increased arterial hypertension), all of * Corresponding author. Division of Pediatric Nephrology, Room No 406, PGIMER Building, PGIMER, Dr RML Hospital, New Delhi 201010, India. Tel.: þ91 9711007064. E-mail address: [email protected] (A. Saha). http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.055 0021-9150/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
these can result in endothelial dysfunction, which is now emerging as the most important component in the thrombotic process [5,6]. Thus endothelial damage may constitute a link between nephrosis and vascular complications. It is a widely held view that impaired endothelial function is the initial step in atherogenesis, which is largely responsible for ischaemic heart disease and thrombotic strokes decade’s later [7]. Soluble thrombomodulin (sTM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), von-Willebrand factor (vWF) are factors disturbed when endothelial cells are damaged, raised levels of these factors have been proposed as markers of endothelial dysfunction [8e10]. These markers reflect the process from endothelial activation to damage and are considered as components involved in complex process of atherosclerotic and thrombotic diseases [11,12]. The current standard of care is not to use therapy with HMG CoA reductase inhibitors (e.g. atorvastatin) in steroid sensitive nephrotic syndrome (SSNS) as hyperlipedimia is considered transient [13]. However there is evidence that statins significantly improve
B. Sharma et al. / Atherosclerosis 233 (2014) 704e706
endothelial dysfunction [14]. There is also evidence that the intimal medial thickness of carotid vessels in adults who had nephrotic syndrome in childhood, depends on the number of relapses [4]. Fifty to sixty percent of children with SSNS have frequently relapsing course. The objective of this study was to measure plasma levels of vWF, t-PA, PAI-1 and sTM in children with first episode of idiopathic nephrotic syndrome (FENS) compared to controls and in drug induced remission.
2. Methods 2.1. Study design and patient groups This was an analytical study with longitudinal follow-up, conducted at a tertiary care teaching hospital in New Delhi, India from June 2011 to October 2012 and was approved by the Institutional Review Board and informed consent was obtained from the legal guardian. Children aged between 1 year and 16 years with FENS were eligible for inclusion in the study. Those with secondary nephrotic syndrome, thromboembolic complications, bleeding diathesis, pre-existing hypertension, diabetes mellitus, blood transfusion, drugs affecting endothelial functions and refusal of informed consent were excluded from the study. Samples for children in all the groups were collected at initial diagnosis and none received second line agents at the time of sampling. We recruited 42 patients with FENS, and 40 age and sex matched healthy controls. FENS was treated with 2 mg/kg of prerdnisolone for 6 weeks followed by 1.5 mg/kg for next 6 weeks. Guidelines recommended by Indian Society of Pediatric Nephrology were used for defining frequently relapsing nephrotic syndrome (FRNS), steroid dependent nephrotic syndrome (SDNS) and steroid resistant nephrotic syndrome (SRNS) [13]. Ultrasound guided percutaneous renal biopsy was performed in all cases of SRNS. Primary outcome measure was levels of sTM, PAI-1, vWF and t-PA in children with FENS and in controls.
2.2. Measurement of endothelial dysfunction markers Plasma was obtained by centrifuging the blood at 2800 g for 10 min and stored immediately in four different aliquots at 80 C until analysed. Quantitative estimation of the markers was done using ELISA technique. Kits were used for the respective markers (vWF, PAI-1, tPA [AssayproÔ LLC USA] and for TM [Gen-Probe Diaclone SAS, France]). The kits contained pre-coated antibodyplates, detection antibodies, buffers, diluents, standards, and substrates. The measurement was done on Evolis Twin Plus machine (Bio-RadÔ) machine. Intra -assay and inter -assay coefficients of variation were 4.7% and 7.2% for PAI-1, vWF and t-PA; for sTM this value was 3.9% and 9.8% respectively.
2.3. Statistical analysis Statistical analysis was done using SPSS software version 16. Continuous variables were presented as the mean standard deviation (SD) or median (interquartile range) while categorical variables were presented as the frequency and percentage. Comparison between independent continuous variables was performed using ManneWhitney U test. Correlations were assessed by Spearmann rank tests. Spearman’s rho “r” correlation coefficient was used to measure the linear relationship between continuous variables. A p-value less than 0.05 were considered statistically significant.
705
3. Results Children with nephrotic syndrome had high serum cholesterol, serum LDL, serum triglycerides, serum VLDL, urinary protein creatinine ratio (p < 0.001). Base line characters were similar in both the groups (Supplementary Table 1). Of the study group (42), 10 were initial SRNS (6 minimal change disease, 4 focal segmental glomerulosclerosis) 28 were infrequent relapsers, 4 patients became FRNS/SDNS at 1 year of follow up. The levels of markers of endothelial dysfunction (sTM, tPA, PAI1, vWF) were significantly higher (p < 0.001) in children with FENS as compared to controls at the onset of nephrotic syndrome (Table 1). Adjusted p values were calculated after log transformation for BMI. There was a significant decrease in the levels of the markers of endothelial dysfunction (sTM, tPA, PAI-1, vWF) at 12 weeks in cases, with treatment with corticosteroids (Table 2). Children with SRNS had raised levels of markers at onset, compared to steroid sensitive patients at onset of nephrotic syndrome. Three markers had significantly raised values (sTM, tPA, vWF (p < 0.05)) where as PAI-1 did not show statistical significant difference (Table 2). Those patients who developed SRNS had no difference in the levels of sTM, tPA, PAI-1 and vWF, at disease onset and at 4 weeks of treatment with steroids. sTM showed a significantly positive correlation with total cholesterol (r ¼ 0.453; p < 0.003) and with low density lipoprotein (r ¼ 0.414; p < 0.006), tPA levels showed a significantly positive correlation with urine protein to creatinine ratio (r ¼ 0.311; p < 0.04) in children with FENS.
4. Discussion Endothelial dysfunction is an early phase of atherogenesis associated with increased levels of the markers of endothelial dysfunction and may constitute a link between nephrotic syndrome and accelerated atherosclerosis. Our results are consistent with the study by Tkaczyk et al., where the levels of sTM, tPA, PAI-1 and vWF were found to be significantly raised in children with INS as compared to controls [15]. Shouman et al. showed that levels of thromboxane, a marker of decreased vasoreactivity, were also significantly raised in children with INS compared to controls [16]. Zhang et al. reported raised levels of vWF, sTM and Malyszko et al. found raised levels of soluble thrombomodulin (sTM) in nephrotic cases as compared to controls [17,18]. This proves that endothelial dysfunction starts at the very onset of nephrotic syndrome in children. The levels of all the markers were found to decrease significantly from the onset to drug induced remission at 12 weeks. However the levels of all the markers were higher in INS group compared to controls, at drug induced remission (12 weeks). These results are consistent with the studies by Tkaczyk et al. and Shouman et al. [16,17]. Hence with remission though, endothelial dysfunction decreases, it does not subside completely at least in the short term.
Table 1 Plasma levels markers of endothelial dysfunction at onset of nephrotic syndrome as compared to controls (whole group). Markers
Nephrotic syndrome (n ¼ 42) Controls (n ¼ 40)
sTM (ng/ml) 19.84 tPA (ng/ml) 3.73 PAI-1 (ng/ml) 27.05 vWF (mU/ml) 1316.11
(8.72e72.54) (2.01e9.28) (18.61e30.42) (992.66e2276.24)
3.95 0.91 3.41 392.07
(2.43e5.05) (0.30e1.21) (1.67e6.13) (375.89e417.58)
P Value
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Endothelial dysfuntion in children with idiopathic nephrotic syndrome Bharat Sharma a, Abhijeet Saha a, *, N.K. Dubey a, Kanika Kapoor a, Anubhuti b, Vinita Vijay Batra c, Ashish Datt Upadhayay d a
Division of Pediatric Nephrology, Department of Pediatrics, Postgraduate Institute of Medical Education & Research, Dr Ram Manohar Lohia Hospital, New Delhi, India Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Dr Ram Manohar Lohia Hospital, New Delhi, India c Department of Pathology, GB Pant Hospital, New Delhi, India d Department of Biostatistics, AIIMS, New Delhi, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2013 Received in revised form 21 January 2014 Accepted 30 January 2014 Available online 11 February 2014
Background: Impaired endothelial function is the initial step in atherogenesis, which is largely responsible for ischaemic heart disease and thrombotic strokes decades later. Methods: Fourty two children with first episode nephrotic syndrome (FENS) aged 1e16 years and 40 controls were enrolled. Soluble thrombomodulin (sTM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and von-willebrand factor (vWF) levels were measured in plasma in FENS, at 12 weeks of drug induced remission and in steroid resistant nephrotic syndrome (SRNS) patients at diagnosis. Results: PAI-1, sTM, vWF and t-PA were significantly raised at the onset of nephrotic syndrome (p < 0.0001). All the markers had a fall after 12 weeks of steroid treatment, but were still raised. Children with SRNS had higher levels of sTM, tPA, vWF as compared to infrequent relapsers, at onset and at 4 weeks of steroid treatment. Conclusion: Children with idiopathic nephrotic syndrome have endothelial dysfunction which is largely dependent upon disease activity. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endothelial dysfunction Nephrotic syndrome Children
1. Introduction Long term studies in adult nephrotic population indicate association of cardiovascular diseases in these patients [1]. Ordonez et al has confirmed that patients with nephrotic syndrome have a 5.5 fold increased relative risk of cardiovascular infarction, resulting in an almost threefold increase in cardiac mortality [2]. Risk factors for early atherosclerosis in adult population include hyperhomocystenemia, proteins of chronic inflammatory process, prothrombotic factors, and adhesion molecules [3]. Many of these risk factors appear in paediatric patients at the time of first occurrence or during aggravation of idiopathic nephrotic syndrome (INS) [4]. Their appearance is related to excessive loss of albumin in urine, oxidative stress, or to steroid therapy (leading to obesity, glucose utilisation disorders, and increased arterial hypertension), all of * Corresponding author. Division of Pediatric Nephrology, Room No 406, PGIMER Building, PGIMER, Dr RML Hospital, New Delhi 201010, India. Tel.: þ91 9711007064. E-mail address: [email protected] (A. Saha). http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.055 0021-9150/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
these can result in endothelial dysfunction, which is now emerging as the most important component in the thrombotic process [5,6]. Thus endothelial damage may constitute a link between nephrosis and vascular complications. It is a widely held view that impaired endothelial function is the initial step in atherogenesis, which is largely responsible for ischaemic heart disease and thrombotic strokes decade’s later [7]. Soluble thrombomodulin (sTM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), von-Willebrand factor (vWF) are factors disturbed when endothelial cells are damaged, raised levels of these factors have been proposed as markers of endothelial dysfunction [8e10]. These markers reflect the process from endothelial activation to damage and are considered as components involved in complex process of atherosclerotic and thrombotic diseases [11,12]. The current standard of care is not to use therapy with HMG CoA reductase inhibitors (e.g. atorvastatin) in steroid sensitive nephrotic syndrome (SSNS) as hyperlipedimia is considered transient [13]. However there is evidence that statins significantly improve
B. Sharma et al. / Atherosclerosis 233 (2014) 704e706
endothelial dysfunction [14]. There is also evidence that the intimal medial thickness of carotid vessels in adults who had nephrotic syndrome in childhood, depends on the number of relapses [4]. Fifty to sixty percent of children with SSNS have frequently relapsing course. The objective of this study was to measure plasma levels of vWF, t-PA, PAI-1 and sTM in children with first episode of idiopathic nephrotic syndrome (FENS) compared to controls and in drug induced remission.
2. Methods 2.1. Study design and patient groups This was an analytical study with longitudinal follow-up, conducted at a tertiary care teaching hospital in New Delhi, India from June 2011 to October 2012 and was approved by the Institutional Review Board and informed consent was obtained from the legal guardian. Children aged between 1 year and 16 years with FENS were eligible for inclusion in the study. Those with secondary nephrotic syndrome, thromboembolic complications, bleeding diathesis, pre-existing hypertension, diabetes mellitus, blood transfusion, drugs affecting endothelial functions and refusal of informed consent were excluded from the study. Samples for children in all the groups were collected at initial diagnosis and none received second line agents at the time of sampling. We recruited 42 patients with FENS, and 40 age and sex matched healthy controls. FENS was treated with 2 mg/kg of prerdnisolone for 6 weeks followed by 1.5 mg/kg for next 6 weeks. Guidelines recommended by Indian Society of Pediatric Nephrology were used for defining frequently relapsing nephrotic syndrome (FRNS), steroid dependent nephrotic syndrome (SDNS) and steroid resistant nephrotic syndrome (SRNS) [13]. Ultrasound guided percutaneous renal biopsy was performed in all cases of SRNS. Primary outcome measure was levels of sTM, PAI-1, vWF and t-PA in children with FENS and in controls.
2.2. Measurement of endothelial dysfunction markers Plasma was obtained by centrifuging the blood at 2800 g for 10 min and stored immediately in four different aliquots at 80 C until analysed. Quantitative estimation of the markers was done using ELISA technique. Kits were used for the respective markers (vWF, PAI-1, tPA [AssayproÔ LLC USA] and for TM [Gen-Probe Diaclone SAS, France]). The kits contained pre-coated antibodyplates, detection antibodies, buffers, diluents, standards, and substrates. The measurement was done on Evolis Twin Plus machine (Bio-RadÔ) machine. Intra -assay and inter -assay coefficients of variation were 4.7% and 7.2% for PAI-1, vWF and t-PA; for sTM this value was 3.9% and 9.8% respectively.
2.3. Statistical analysis Statistical analysis was done using SPSS software version 16. Continuous variables were presented as the mean standard deviation (SD) or median (interquartile range) while categorical variables were presented as the frequency and percentage. Comparison between independent continuous variables was performed using ManneWhitney U test. Correlations were assessed by Spearmann rank tests. Spearman’s rho “r” correlation coefficient was used to measure the linear relationship between continuous variables. A p-value less than 0.05 were considered statistically significant.
705
3. Results Children with nephrotic syndrome had high serum cholesterol, serum LDL, serum triglycerides, serum VLDL, urinary protein creatinine ratio (p < 0.001). Base line characters were similar in both the groups (Supplementary Table 1). Of the study group (42), 10 were initial SRNS (6 minimal change disease, 4 focal segmental glomerulosclerosis) 28 were infrequent relapsers, 4 patients became FRNS/SDNS at 1 year of follow up. The levels of markers of endothelial dysfunction (sTM, tPA, PAI1, vWF) were significantly higher (p < 0.001) in children with FENS as compared to controls at the onset of nephrotic syndrome (Table 1). Adjusted p values were calculated after log transformation for BMI. There was a significant decrease in the levels of the markers of endothelial dysfunction (sTM, tPA, PAI-1, vWF) at 12 weeks in cases, with treatment with corticosteroids (Table 2). Children with SRNS had raised levels of markers at onset, compared to steroid sensitive patients at onset of nephrotic syndrome. Three markers had significantly raised values (sTM, tPA, vWF (p < 0.05)) where as PAI-1 did not show statistical significant difference (Table 2). Those patients who developed SRNS had no difference in the levels of sTM, tPA, PAI-1 and vWF, at disease onset and at 4 weeks of treatment with steroids. sTM showed a significantly positive correlation with total cholesterol (r ¼ 0.453; p < 0.003) and with low density lipoprotein (r ¼ 0.414; p < 0.006), tPA levels showed a significantly positive correlation with urine protein to creatinine ratio (r ¼ 0.311; p < 0.04) in children with FENS.
4. Discussion Endothelial dysfunction is an early phase of atherogenesis associated with increased levels of the markers of endothelial dysfunction and may constitute a link between nephrotic syndrome and accelerated atherosclerosis. Our results are consistent with the study by Tkaczyk et al., where the levels of sTM, tPA, PAI-1 and vWF were found to be significantly raised in children with INS as compared to controls [15]. Shouman et al. showed that levels of thromboxane, a marker of decreased vasoreactivity, were also significantly raised in children with INS compared to controls [16]. Zhang et al. reported raised levels of vWF, sTM and Malyszko et al. found raised levels of soluble thrombomodulin (sTM) in nephrotic cases as compared to controls [17,18]. This proves that endothelial dysfunction starts at the very onset of nephrotic syndrome in children. The levels of all the markers were found to decrease significantly from the onset to drug induced remission at 12 weeks. However the levels of all the markers were higher in INS group compared to controls, at drug induced remission (12 weeks). These results are consistent with the studies by Tkaczyk et al. and Shouman et al. [16,17]. Hence with remission though, endothelial dysfunction decreases, it does not subside completely at least in the short term.
Table 1 Plasma levels markers of endothelial dysfunction at onset of nephrotic syndrome as compared to controls (whole group). Markers
Nephrotic syndrome (n ¼ 42) Controls (n ¼ 40)
sTM (ng/ml) 19.84 tPA (ng/ml) 3.73 PAI-1 (ng/ml) 27.05 vWF (mU/ml) 1316.11
(8.72e72.54) (2.01e9.28) (18.61e30.42) (992.66e2276.24)
3.95 0.91 3.41 392.07
(2.43e5.05) (0.30e1.21) (1.67e6.13) (375.89e417.58)
P Value
