Introduction

The generation of reactive oxygen species (ROS) is a normal steady state process in cells, although uncontrolled production results in damage to cellular molecules. Malondialdehyde (MDA), the main indicator of lipid peroxidation, causes the cross linking and polymerization of membrane components and also reacts with DNA nitrogenous bases [1]. Cellular defense mechanisms against ROS include enzyme systems that directly remove these species (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic scavengers that are either endogenous molecules (albumin, glutathione and uric acid) or derived from the diet (vitamin C, vitamin E, carotenoids, selenium and zinc). The cumulative effect of endogenous and food-derived antioxidants represents the total antioxidant capacity (TAC) of the system. An imbalance between the production and elimination of ROS is referred to oxidative stress [2].

The study of oxidant/antioxidant balance in primary nephrotic syndrome (PNS) in both animal [3, 4] and human [5–7] models has gained the attention of many researchers in recent years. However, the results of these studies have been conflicting. Almost all of the human studies were carried out on patients with steroid-sensitive nephrotic syndrome. Moreover, whether the studied patients were relapsers or non-relapsers was not taken into account. The longitudinal study reported here was carried out to assess the serial changes in serum TAC and MDA levels in newly diagnosed PNS children. We also evaluated the prognostic value of TAC and MDA levels in predicting the response to corticosteroids.

Subjects and methods

Subjects

This study was a prospective observational study carried out on 33 newly diagnosed children with PNS. The patients were consecutively recruited from the Nephrology Unit at Mansoura University Children's Hospital during the period from February 2005 to October 2006. They included 22 male and 11 female pediatric patients whose ages ranged from 2 to 10 years. Ten healthy children of matched age and sex were included in the study as a control group.

Patients were diagnosed to have PNS according to the criteria of the International Study of Kidney Disease in Children (ISKDC) [8]. None of the children enrolled in the study showed evidence of acute infection or systemic diseases, and none had received albumin, blood or non-steroidal anti inflammatory drugs in the last 2 weeks prior to the study.

All patients received standard oral corticosteroids induction therapy for 1 month. If the patients achieved remission, the corticosteroids were tapered and withdrawn over a 2-month period.

Serum levels of TAC and MDA were measured at three stages of the disease:

  1. Stage I:

    (proteinuric stage): the period immediately following the diagnosis of PNS in all patients, prior to the initiation of corticosteroid treatment;

  2. Stage II:

    (remission on corticosteroids): at the end of the induction of corticosteroid therapy in patients who achieved remission;

  3. Stage III:

    (after weaning of corticosteroids): immediately after standard corticosteroid therapy had been stopped.

Patients were followed up until the last clinic visit (December 2008).

The patients were retrospectively classified into two groups according to the criteria of ISKDC [8]: steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS). The SSNS group (n = 26) included those who responded to standard corticosteroid induction therapy, of whom 15 were non-relapsers (patients who did not show any relapse during the follow-up period) and 11 were relapsers (patients who developed one or more relapses). The SRNS group (n = 7) comprised patients who did not respond to standard corticosteroid induction therapy. Renal biopsy of these patients revealed focal segmental glomerularsclerosis in three patients, minimal change nephrotic syndrome in two patients and diffuse mesangial proliferation in two patients.

Measurement of serum TAC Levels

Serum TAC levels were measured according to the method described by Koracevic et al. [9] in which the determination of antioxidative capacity is based on the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide. The antioxidants in the sample eliminate a certain amount of the provided hydrogen peroxide, and the residual hydrogen peroxide is determined calorimetrically by an enzymatic reaction that involves the conversion of 3,5 dichloro-2-hydroxy benzensulphonate to a colored product.

Measurement of serum MDA

The colorimetric method described by Satoh [10] and Ohkawa et al. [11] was used to measure serum MDA levels. Thiobarbituric acid reacts with MDA in an acidic medium at 95°C for 30 min to form a thiobarbituric acid reactive product. The absorbance of the resultant pink product can be measured at 534 nm.

Statistical analysis

The SPSS software program, ver.10 (SPSS, Chicago, IL) was used to analyze the data. Data on the clinical and laboratory characteristics of patients at presentation are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare these data between patients and controls. The chi-square test was used to assess gender distribution in the studied groups. The serum levels of MDA and TAC are presented as the median and range. The Mann–Whitney test was used to compare data between two groups, and the Wilcoxon Signed Rank test was used to compare one group at different times. A receiver operating characteristic (ROC) curve was drawn to differentiate between SSNS and SRNS as well as between non-relapsers and relapsers. P values ≤ 0.05 were considered to be significant.

Results

Characteristics of the study cohort

The clinical and demographic data on the children enrolled in the study are given in Table 1. The data presented in Table 2 reveal that, relative to the controls, all of the patients enrolled in the study had significantly lower TAC serum levels during the three stages of the disease. In comparison, serum MDA levels were significantly higher in the patient group in stage I and II only, and no significant difference was observed in stage III.

Table 1 Descriptive data of the study cohort at presentation
Full size table
Table 2 Comparison of serum total antioxidant capacity and malondialdehyde levels between the studied patients and controls at different stages of primary nephrotic syndrome
Full size table

In stage I, SRNS patients had significantly lower TAC levels (P = 0.001) and higher MDA levels (P = 0.003) than SSNS patients (Table 3).

Table 3 Comparison of serum levels of TAC and MDA between SSNS and SRNS patients in the proteinuric stage of primary nephrotic syndrome
Full size table

Figure 1a demonstrates that TAC levels significantly increased in both non-relapsers (P = 0.001) and relapsers (P = 0.005) in stage II compared to stage I. A further significant increase was observed in stage III in non-relapsers (P = 0.001) and relapsers (P = 0.003). No significant difference in TAC levels was observed between non-relapsers and relapsers in stage I. However, non-relapsers had higher TAC levels in stages II (P = 0.004) and III (P = 0.001) than relapsers.

Fig. 1
figure 1

Serial changes in serum total antioxidant capacity (TAC) (a) and malondialdehyde (MDA) level (b) in relapsers and non-relapsers at different stages of primary nephrotic syndrome (PNS)

Full size image

There were no significant differences in MDA levels between stage I and II in both relapsers and non-relapsers, but MDA levels were significantly lower in stage III than stage II in both non-relapsers (P = 0.001) and relapsers (0.004). No significant differences were observed in MDA levels between non-relapsers and relapsers in the three stages of PNS (Fig. 1b).

Figure 2 shows that it is possible to predict the response to corticosteroids in the proteinuric stage of PNS by using TAC levels at the cut-off point ≥ 0.73 mM/L (sensitivity 89%, specificity 86%, predictability 93%). Alternatively, TAC levels after the weaning of corticosteroids at a cut-off point ≤1.14 mmol/L could be used to predict relapses (sensitivity 91%, specificity 80%, and predictability 88%), as presented in Fig. 3.

Fig. 2
figure 2

The receiver operating characteristic (ROC) curve to differentiate between steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS) in the proteinuric stage of PNS using serum TAC levels

Full size image
Fig. 3
figure 3

The ROC curve to differentiate between non-relapsers and relapsers using serum TAC after the weaning of corticosteroids

Full size image

Discussion

To the best of our knowledge, this is the first study to assess serial changes in oxidants and antioxidants in a cohort of newly diagnosed children with PNS with different responses to corticosteroids.

All of the newly diagnosed patients with PNS in the proteinuric phase of the disease prior to starting corticosteroid therapy had oxidative stress in the form of high serum MDA with low TAC concentrations. This condition of oxidative stress was similar whether the patients were proved later to have SRNS or SSNS (either non-relapsers or relapsers). Interestingly, this pattern was more evident in SRNS than in SSNS, while it was similar in non-relapsers and relapsers.

Low TAC concentrations have been reported in the proteinuric phase of SSNS during the first episode [12, 13] or at relapse [13]. In contrast, Karthikeyan et al. reported significantly increased ferric-reducing antioxidant power in nephrotic syndrome in the active disease state and that this remained high during remission when compared to the controls [14]. While many studies have documented high serum MDA levels [15–17], Ginveri et al. [6] reported high intra-erythrocyte concentrations of MDA and reduced concentrations of the antioxidant glutathione in both SSNS and SRNS. However, Gusmano et al. [18] and Kinra et al.[19] observed an insignificant rise of serum MDA, which they explained as being due to factors associated with the study design, including small sample size and bias resulting from their selection of hospital controls, as well as to the possibility that the much higher concentrations of membrane cholesterol found in the acute phase provided a protective effect.

The low TAC concentration in the proteinuric phase of PNS could be attributable to many mechanisms. Hypoalbuminemia may be a contributory factor as albumin is the leading preventive antioxidant of the serum due to its role as a metal binding protein [20]. Other contributory factors are the impaired intestinal absorption of some antioxidant components and/or the low antioxidant content of the diet in patients with PNS as most have a poor appetite [21]. There are some data in the literature showing that a diet deficient in selenium and vitamin E may lead to renal injury characterized by proteinuria [22]. The low TAC during the acute stage may also result from the increased consumption of certain antioxidant components, such as vitamin C, which is significantly lower in the acute phase of nephrotic syndrome [7]. The impaired antioxidant status enhances lipid peroxidation. There is some evidence available showing that during the proteinuric stage of PNS circulating lipids bind to and become trapped by extracellular matrix molecules where they undergo oxidation, thereby increasing the formation of ROS [23]. Enhanced lipid peroxidation is known to be manifested by increased serum MDA levels [1].

During remission when patients were still receiving corticosteroids, serum TAC improved in both non-relapsers and relapsers compared to the proteinuric stage; however, the levels were still significantly lower than those found in the controls. This improvement was more marked in non-relapsers than in relapsers. In comparison, although serum MDA levels were still significantly higher in patients during remission than in controls, they did not significantly differ from levels in the proteinuric stage, and there was no significant difference between non-relapsers and relapsers. Previous studies have reported similar changes in serum TAC [12] and MDA [13, 24] concentrations in patients with SSNS during the remission stage. High serum MDA levels were more common among the relapsers than in another group of patients during remission [24], but none of these earlier studies took the differences between non-relapsers and relapsers into account.

The normalization of serum albumin and the improvement in a patient’s appetite as well as intestinal absorption of antioxidants may be responsible for the increase in TAC levels during the remission of the disease. The use of corticosteroids may be another explanation for the improvement of TAC levels. In an experimental rat model, the administration of corticosteroids increased glomerular antioxidant enzymes, which in turn reduced the proteinuria caused by hydrogen peroxide infusion [25].

There was a further improvement in serum TAC levels in both non-responders and responders following weaning of the corticosteroids, in comparison to the previous two stages of the disease, but the levels were still lower than those found in the controls. Interestingly, relapsers still had lower TAC levels than non-relapsers. In contrast, the MDA level decreased significantly in both groups and reached the level found in the controls; no significant difference was observed between non-relapsers and relapsers.

The persistence of oxidative stress in patients with SSNS after long-term remission has been reported by a number of investigators [26, 27]. These changes in the studied parameters suggest that although there is clinical remission, as indicated by the absence of proteinuria and the normalization of serum albumin, ongoing oxidative stress still remains, even during remission. This indicates that the body can withstand a certain degree of stress, only beyond which do proteinuria and the clinical picture of nephrotic syndrome occur [15].

It is difficult to interpret why patients with SRNS had more oxidative stress than SSNS patients at the time of diagnosis and why the oxidative stress was more evident in relapsers than in non-relapsers during the remission of the disease. It can be speculated that the oxidant/antioxidant imbalance partly modulates the response of newly diagnosed PNS patients to corticosteroids. Since our study cohort was insufficient in terms of the number of enrolled patients, further studies with larger numbers of patients are required to prove/disprove this hypothesis. However, our results are encouraging in terms of suggesting the use of serum TAC and MDA levels as potential predictors of the response of newly diagnosed patients to corticosteroid therapy. At the time of diagnosis, we found that the response to corticosteroids could be predicted at serum TAC level ≥0.73 mM/L (sensitivity 89%, specificity 86%), while serum TAC levels ≤1.14 mM/L after the weaning of corticosteroids could predict that the patient would be a relapser (sensitivity 91%, specificity 80%).

In conclusion, newly diagnosed patients with PNS persistently have oxidant stress even during remission of the disease. This stress may modulate the response to corticosteroids. Serum TAC level at the time of the diagnosis could predict the response to corticosteroids while serum TAC after weaning of corticosteroids could predict whether the patient would relapse or not. Further prospective studies using larger numbers of patients are needed to validate these results.