Main

Minimal change nephrotic syndrome (MCNS) is the most common form of nephrotic syndrome (NS) in children and generally responds well to treatment with prednisone (1). An important advance in understanding the pathogenesis of NS was the observation that oxygen-free radicals are possible mediators of injury in experimental nephrosis in rats. The similarity of that animal model to human MCNS provokes the idea that free-radical mediated injury could play a role in the pathogenesis of that disorder (2). This is probably a consequence of an imbalance between oxidant and antioxidant activity in vivo (3). The injection of antioxidants in experimental models of NS has been found to be ameliorative (4). It has been suggested that enhanced permeability of the glomerular capillary wall is possibly influenced by the generation of free radicals (5). Furthermore, dyslipidemia of NS is, also, known to be linked to oxidative reactions and atherosclerosis (6,7). Patients with NS have elevated concentrations of total cholesterol (TC), LDL cholesterol (LDL-C), and triglycerides (TG), whereas, HDL cholesterol (HDL-C) has variously been reported to be increased, decreased, or normal (7). These abnormalities suggest that nephrotic patients have a higher risk in developing atherosclerosis (8). Both the lipid and the protein moieties of LDL-C are subject to oxidation and both can be oxidized in vivo. Oxidation of LDL-lipid moiety (oxLDL) is commonly thought to represent the initial step of oxidative LDL modification. The oxidative modification of LDL may represent an important pathway in the pathogenesis of atherogenesis (9). Direct assessment of reactive oxygen species (ROS) is not feasible because of the extremely short half-life of the free radicals (6). Therefore, the oxidative activity must be measured indirectly by the levels of lipid membranes peroxidation by-product; malondialdehyde (MDA). Also, by measuring the levels of antioxidant substances e.g., glutathione peroxidase (GSH-PX) (E.C.I.11.1.9) which is one of the most important scavenging enzymes (10), antioxidant vitamins (vitamins C, A, and E) and albumin which is a nonenzymatic antioxidant protein.

The present study aimed to assess oxidant (in terms of serum levels of MDA and oxLDL) and antioxidant status (in terms of serum levels of albumin, GSH-PX, and vitamins C, A, and E) in relation to dyslipidemia [in terms of serum levels of TC, LDL-C, HDL-C, TG, apolipoprotein A-I (apo-A-I), and apolipoprotein-B (apo-B)] in children during remission and relapse phases of steroid sensitive nephrotic syndrome (SSNS).

METHODS

The present study was conducted on 40 children (22 boys/18 girls) diagnosed as SSNS. They were admitted to Pediatric University Hospital, Assiut University. Their ages ranged 5–9 y. They were categorized into two subgroups. The first subgroup included 25 children during remission stage (after 6 wk of regular dose 2 mg/kg/d of prednisolone therapy alone). Their age (mean ± SD) was 7.16 ± 0.95 y. The second subgroup included 15 children during relapse (8 infrequent relapsers: patients who developed relapse once/y and 7 frequent relapsers: patients who developed relapse three times or more/y). Their age (mean ± SD) was 6.66 ± 1.11 y. Kidney biopsy was not performed for the studied patients.

Exclusion criteria were children with (i) Steroid resistant NS and NS secondary to renal involvement of systemic diseases; (ii) acute infection; (iii) features suggestive of chronic renal failure; and (iv) clinical and anthropometric features of malnutrition and/or signs of vitamin deficiency. In addition, a control group of 15 healthy age (mean ± SD, 7.43 ± 1.19 y) and gender (9 boys/6 girls) matched children were included. All the studied groups belong to a homogenous population with no difference in their cultural or socioeconomic status (to ensure minimal nutritional differences between the studied groups).

An informed written consent was obtained from the parents of each subject and the Ethics Committee of the Faculty of Medicine at Assiut University approved the study.

After an overnight fast, 5 mL venous blood was collected from each subject. The blood was allowed to clot at room temperature and centrifuged at 5000 rpm for 10 min, and then the serum was collected and kept frozen at −70°C in aliquots until the time of assay of the following biochemical parameters.

Serum MDA was estimated according to the method described by Buege and Aust (11). Oxidized LDL was estimated by using the Immulisa anti-oxLDL antibody ELISA kit (Cat. No.1158; IMMCO Diagnostic, Inc., NY) (12). Serum albumin was determined colormetrically by a commercial kit supplied by Spectrum (ALB 080) Egypt (13). Serum GSH-PX activity was determined according to the method of Mills (14) and as modified by Hafeman et al., (15). Vitamin C was determined by the 2, 4 dinitrophenylhydrazin method of Roe (16). The method used for vitamin A determination was described by Nino and Parasad (17). Vitamin E (tocopherols) was determined according to the method of Baker et al., (18). Serum levels of LDL-C were determined as the difference between TC and the cholesterol contents of the supernatant fluid after precipitation of the LDL fraction by polyvinyl sulfate (Quimica Clinica Aplicada, Spain) (19). Serum HDL-C was determined colormetrically by using Stanbio HDL-C kit (Cat. No. 6599; Quimica Clinica Aplicada, Spain) (20). TC and TG serum levels were measured by colorimetric method using kit supplied by Stanbio Laboratory Inc. [Cat. No. 1010-430 (21) and 2100-430 (22), respectively]. Serum apo-A-I and apolipoprotein B were determined by radial immunodiffusion plates (Diffu-plate) provided by Biosentifica, Buenos Aires, Argentina (23).

Statistical analysis.

The statistical analysis was done using Statistical Package for Social Science (SPSS) version 12. Data were expressed as mean ± SD. Normally distributed data were analyzed using student t-test whereas abnormally distributed data (skewed) were statistically analyzed using Mann Whitney test. Spearman correlation coefficient between measured parameters was evaluated by the statistical significance of their linear regression. p value of less than 0.05 is considered significant.

RESULTS

The serum levels (mean ± SD and range) of MDA and oxLDL in controls and children with SSNS at remission and relapse are presented in Table 1. Significant higher levels of MDA and oxLDL were observed in children with SSNS at both remission and relapse in comparison with those of the controls (p < 0.001), with significant higher levels (p < 0.001) in relapse group in comparison with those of the remission group. Table 2 demonstrates the serum levels (mean ± SD and range) of albumin, GSH-PX activity, and vitamins C, A, and E in controls and in children with SSNS at remission and relapse. Children at remission showed significant lower levels of GSH-PX activity (p < 0.001), vitamin-C (p < 0.001), vitamin-A (p < 0.001), and vitamin-E (p < 0.05) in comparison with those of the controls, except for albumin, which showed no significant difference. The relapse group showed significantly lower levels of the studied bioindices in comparison with those of the controls (p < 0.001) and remission (p < 0.001). The serum levels of TC, LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglycerids (TG), apo-A-I (apo A-I), and apolipoprotein B (apo-B) in controls and children with SSNS at remission and relapse are presented in Table 3. Both the studied groups of SSNS children showed significant higher levels of the studied parameters in comparison with those of the controls, except HDL-C, whose mean levels in both the SSNS groups were significantly lower than the controls. Children at relapse showed significantly higher mean levels of TC, LDL-C, and TG and significantly lower mean levels of HDL-C in comparison with those of the remission group (p < 0.001), except for apo-A-I and apo-B levels that showed no significant difference. Correlations among various studied bioindices in children with SSNS are presented in Table 4. Significant positive correlations were observed between serum levels of MDA, oxLDL, TC, LDL-C, apo-A-I, and apo-B. In addition, each of the MDA and oxLDL levels were significantly negatively correlated with albumin, GSH-PX, vitamins A, C, E, and HDL-C.

Table 1 Serum levels of malondialdehyde (MDA) and oxidized LDL in controls and children with steroid sensitive nephrotic syndrome at remission and relapse
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Table 2 Serum levels of albumin, glutathione peroxidase activity and vitamins C, A, and E in controls and in children with steroid sensitive nephrotic syndrome at remission and relapse
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Table 3 Serum levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides (TG), apolipoprotein A-I (apo A-I) and apolipoprotein B (apo-B) in A and E in controls and children with steroid sensitive nephrotic syndrome at remission and relapse
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Table 4 Correlation coefficient (r) among various bioindices in children with steroid sensitive nephrotic syndrome
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DISCUSSION

In the last few years, it has been proved that atherosclerotic changes begin to develop in childhood (24). Moreover, some clinical reports claim a high incidence of ischemic heart failure in adult nephrotic patients (8). Because the life expectancy of children and adolescents afflicted with NS has dramatically improved over the last 15 y, the occurrence of dyslipidemia with its associated morbidity is of particular concern (25).

Dyslipidemia is a contributory factor in the progression of initial glomerular injury in NS (26). An increase in peroxidation of lipids and lipoproteins may cause the release of ROS, which are strong oxidants that could result in proteinuria and glomerular injury via various mechanisms (27). These ROS can be generated either in the circulation or locally by glomerular cells. They promote cell injury by lipid peroxidation, which disrupts the structural integrity of the tubular epithelial cells and increases the glomerular permeability to proteins together with an alteration in glomerular hemodynamics (6).

The present study investigated the oxidant MDA and the antioxidants; albumin, GSH-PX, and vitamins C, A, and E, together with oxLDL and lipid profile in remission and relapse phases of SSNS (Tables 1–3). Significantly higher mean levels of MDA and lower mean levels of the various antioxidants were observed in SSNS children particularly in those at relapse phase. These findings are consistent with many previous studies (1,3,6,28,29). Peroxidation of lipid membranes raises the concentration of the by-product MDA that results in lowering of the concentration of antioxidants because of consumption (6). Albumin is a leading preventive but not a chain breaking antioxidant of serum. In the present study, significantly lower levels of mean serum albumin were observed in relapse phase in comparison with those of the controls and remission phase. It is reported that even at very low concentrations, albumin has a high antioxidant activity (1). GSH-PX is a selenium-dependent enzyme thought to be critical in intracellular antioxidant defenses (30). Significantly lower mean serum levels of GSH-PX activity were found in SSNS particularly at relapse phase. This low GSH-PX activity levels may be a factor limiting the antioxidant capacity in NS (28,31). Mean serum levels of antioxidant vitamins C, A, and E were significantly decreased in SSNS children especially in relapse phase (Table 2). These decreased levels suggest depletion, possibly because of consumption for neutralizing excessive circulating oxidants (3). Moreover, this could explain the significant negative correlations observed between MDA and the studied antioxidants (Table 4).

The lipids and apolipoprotein profile of SSNS children of the present study have revealed a pattern of abnormalities demonstrated by the significant increased mean serum levels of TC, LDL-C, TG, apo-A-I and apo-B, together with the significant decreased levels of HDL-C in both groups of SSNS children in comparison with those of the controls (Table 3). Significantly higher mean levels were observed in relapse group in comparison with those of the remission group, except for apo-A-I, apo-B, which showed no significant difference between remission and relapse, and HDL-C which was significantly lower in relapse group compared with remission group. These findings agree with previous reports (8,26,32–35). Although dyslipidemia is a common complication of NS, it is unclear whether it is a direct consequence of proteinuria or whether it arises secondarily from other effects of proteinuria such as reduced plasma oncotic pressure or hypoalbuminemia (7,34). Although the observed negative correlation between serum albumin and lipid parameters in patients of the present study (Table 4) was described more than 40 y ago (36), no studies have clearly established a clear causal relationship between the rate of albumin synthesis and dyslipidemia in nephrotic patients (26). The increased levels of serum TC could be attributed to impairment of metabolism of mevalonate (cholesterol precursor) by the nephrotic kidney. This allows a greater cholesterol availability, that coupled with an enhanced hydroxy methyl glutaryl-CoA (HMG-CoA) reductase activity leads to increased hepatic cholesterol synthesis and unbalanced lipid homeostasis (26). In addition, the decreased activity of the enzyme lecithine-cholesterol acyltransferase reported in NS could account for the several lipoprotein abnormalities observed in that syndrome (37). Elevation of LDL-C in NS has been suggested to be caused by at least two independent mechanisms, impairment of LDL-C catabolism because of reduced LDL receptor activity secondary to intrahepatic cholesterol expansion. The impairment of LDL catabolism may, also, be because of a defective ligand-receptor interaction through alteration of the conformation of apo-B on the particle surface (38). The other mechanism is the increased activity of cholesterol ester transfer protein (CETP) [an enzyme that catalyzes the exchange of cholesterol ester rich core of HDL2 for the triglyceride rich core of very LDL (VLDL) remnant particles yielding LDL] in nephrotic patients, thus accelerating the normal pathway for LDL synthesis (33).

In addition, significant higher mean serum levels of oxidized LDL were observed in both the studied groups of SSNS children in comparison with those of the controls, with significant higher levels in relapse group (Table 1). The increased serum levels of oxLDL in NS could be attributed to the significant disturbances in oxidant status during NS (29). OxLDL is commonly thought to represent the initial step of oxidative LDL modification (9). Studies in the last few years have suggested that oxLDL may be critically involved in the progression of glomerulosclerosis. Moreover, Lee et al., (39) reported that LDL oxidation might have a fundamental role in the formation of early atherosclerotic lesions and their progression. This could constitute an important link between NS and atherosclerosis (29).

As regards TG, significantly higher mean serum levels of TG were observed in both the groups of the studied patients particularly in those with relapse (Table 3). Hypertriglyceridemia in the NS is primarily a result of decreased catabolism confirming the observation of Yoshino et al. (40) that it is not caused by increased VLDL secretion. In addition, the decreased lipoprotein catabolism in NS, also caused by decreased clearance of VLDL resulted both from decreased binding of lipoprotein lipase (LpL) to endothelial surfaces in the presence of a reduced oncotic pressure and/or hypoalbuminemia and from an alteration of VLDL binding to endothelium-bound LpL (41). These combined defects may act together to cause the profound defect in lipoprotein catabolism responsible for hypertriglyceridemia in NS (26,34). The influence of NS on HDL-C metabolism is still unclear; decreased as well as normal or elevated HDL-C levels were reported in previous studies (7,8,32). The present study showed significantly lower mean serum levels of HDL-C in SSNS children particularly in those in relapse in comparison with those of the controls and remission (Table 3). HDL-C is an effective antioxidant with the capacity to inhibit the oxidative modification of LDL. HDL-C, also, possesses anti-inflammatory properties. These antioxidant and anti-inflammatory properties of HDL-C may be as important as its cholesterol efflux function in terms of protecting against the development of atherosclerosis (42). Abnormally high excretion of HDL through the damaged glomeruli could be a possible explanation for the reduced serum HDL-C observed in NS (43).

The present study also revealed significantly increased mean serum levels of apo-A-I and apo-B in SSNS children in comparison with those of the controls, with no significant differences between remission and relapse group (Table 3). These findings agree with those of Merouani et al., (8), Kaysen and de Sain-van der Velden (33), and Marsh et al. (38). However, the apo-A-I findings disagree with those of Abdel Ghany et al., (32) and Zhang et al., (44), as they reported decreased levels of apo-A. However, the increased apo-A-I and apo-B in NS could be explained by the hypothesis that changes in plasma oncotic pressure following hypoalbuminemia and may be involved in stimulating the transcription of a group of liver secreted proteins (38,45).

The findings of the present study showed that there is increased oxidative stress during NS with improvement during remission as evidenced biochemically by lower levels of MDA and oxLDL, higher levels of the studied antioxidants and lower levels of lipid profile than those of the relapse group. However, despite the use of glucocorticoids, no normalization of the studied bioindices was observed in remission. The exact mechanism of action of prednisolone in NS has not been elucidated yet, although it is believed to be caused by immunosuppressive and anti-inflammatory properties (3). Moreover, the results of some experimental studies suggest that steroids directly or indirectly impair the antioxidant reactions and lead to overproduction of ROS (46,47). However, the study of Kawamura et al. (48) suggested that glucocorticoids may have a therapeutic role through the stimulation/augmentation of the activity of antioxidant enzymes. In addition, the study of Ece et al. (1) observed that steroid treatment did not suppress oxidative stress in SSNS.

In conclusion, the findings of the present study display an increased oxidative stress and decreased antioxidant response in SSNS. Additionally, although there is clinical remission no normalization of the biochemical indices was observed despite the use of glucocorticoids. Therefore, there might be a potential role for regular lipid monitoring during the follow-up of nephrotic patients especially those with frequent relapses to identify high-risk patients, who should be evaluated as candidates for a lipid lowering therapy. Therefore, the present study recommends the combined use of steroid, antioxidant therapy, and lipid lowering therapy in such children.