MATERIALS AND METHODS

Materials: The present study included 60 children (2-15 years of age) with SRNS in activity constituting the study group and 20 age and sex matching healthy controls. All patients had primary steroid resistance with failure to achieve remission after 6 weeks of daily corticosteroid therapy (60 mg/m²/day) and were thus medically indicated for renal biopsy. Patients with congenital or secondary forms of nephrotic syndrome and those with elevated serum creatinine were excluded. Patients were enrolled from the Nephrology Clinic and in Patient Departments of Cairo University pediatric Hospitals following informed parent consent during the period of May 2007 till November 2008. The study was approved by the Pediatrics Department Research Committee.

Methods: All patients were subjected to:

Ultrasound-guided core renal biopsy after exclusion of any contraindications. In addition to routine light microscopy, unstained slides from the same specimens were further processed in Medical Biochemistry Department, Cairo University. This was done through identification of apoptotic bodies by DNA in situ using TACS. XLTM in situ apoptosis detection kit (R and D system, USA).

Serum MMP-9 and urinary annexin V excretion were measured in control subjects for comparison.

Data analysis: Nominal data were expressed as frequency and percentage, while numerical data were expressed as mean and standard deviation. Means were compared using ANOVA and paired sample t-tests, while proportions were compared using Chi square test. Statistical analysis was done using Microsoft Excel 2003 and SPSS statistical package version 11. p-values less than 0.05 were considered significant.

RESULTS AND DISCUSSION

Pathologically, 35 patients (58%) had focal segmental glomerulosclerosis (FSGS), 19 (32%) had Diffuse Mesangial Proliferation (DMP) and 6 (10%) had minimal change nephropathy (Table 1).

The mean± SD MMP-9 concentration was 87± 53.7 pg mL^-1 in control subjects and 189.1± 150.6 pg mL^-1 in patients before treatment with cyclophosphamide (p<0.0001). After treatment, the mean MMP-9 concentration increased to 329.2± 172.4 pg mL^-1 (p<0.0001) (Fig. 1). Regarding urinary annexin V, the mean control value was 2.7± 0.6 ng g^-1 creatinine. Patients had significantly higher values; 4.5± 0.3 ng g^-1 (p<0.0001), which further increased after treatment (5.2± 1.3 ng g^-1, p = 0.0002).

Table 1:	Clinical data of the study group
*: Data expressed as No. of subjects (%) except for age (Mean± SD). †: Not Significant (p>0.05). SRNS: Steroid-Resistant Nephrotic Syndrome. BP: Blood Pressure

Table 2:	Response to cyclophosphamide and laboratory findings of the study group according to pathological type
Numerical data were expressed as Mean± SD. FSGS: Focal Segmental Glomerulosclerosis, DMP: Diffuse Mesangial Proliferation, MCNS: Minimal Change Nephrotic Syndrome, MMP-9: Matrix Metalloproteinase-9

Fig. 1:	Mean serum matrix metalloproteinase-9 (MMP-9) concentrations in control subjects, patients with steroid-resistant nephrotic syndrome before and after treatment with cyclophosphamide

Fig. 2:	Photomicrograph of kidney tissue showing positive staining for apoptosis in a case of focal segmental glomerulosclerosis

Twenty nine patients (48%) had positive DNA in situ indicating apoptosis in the glomeruli and infiltrating cells of the interstitium (Fig. 2). Table 2 shows the apoptotic markers in patients with different pathological types.

Table 3:	Comparison of apoptotic markers between responders to CPA and resistant cases
Data expressed as Mean± SD, DNA in situ are expressed as No. (%)

Table 4:	MMP-9 and urinary annexin V according to DNA in situ positivity
Data expressed as Mean± SD. MMP-9: Matrix Metalloproteinase-9

Comparing patients who responded to CPA with resistant cases has shown that responsive cases had lower annexin V excretion and DNA in situ positivity (Table 3). Negative apoptosis by DNA in situ predicted response with 72% sensitivity, 69% specificity and a likelihood ratio of 2.47.

Overall, there was no significant correlation between serum MMP9 and urinary annexin V (p>0.10). Table 4 shows the comparison of MMP-9 and urinary annexin V between patients with positive and negative DNA in situ.

In the present study, serum MMP-9, urinary annexin V and evidence of apoptosis in kidney biopsy specimens were investigated in 60 children with SRNS. Pathologically, 58% of the study population had FSGS. Consistent with present findings, FSGS is known to be more resistant than the other types of idiopathic nephrotic syndrome. All patients received oral cyclophosphamide for three months and 25 children (42%) achieved complete remission while 26 (43%) were resistant. This comes in agreement with Ehrich et al. (2007). who stated that the rate of complete remission after induction therapy for SRNS due to either FSGS or Minimal Change Nephrotic Syndrome (MCNS) was below 50%. Meanwhile, Hodson and Craing et al. (2008) stated that ciclosporin (with or without alternate-day prednisone) and cyclophosphamide (with pulse intravenous corticosteroids) resulted in comparable complete or partial remission rates of about 60%.

Apoptosis plays a central role in maintaining homeostasis in the kidney by deletion of unwanted cells without induction of an inflammatory reaction. More than half of the children with SRNS were positive for apoptosis by DNA in situ detection. Those with FSGS had significantly higher rates of apoptosis (69%) compared to those with DMP or MCNS (p = 0.0008). Explanation resides in the fact that apoptosis is highly integrated in the pathogenesis of sclerotic lesions. Schiffer et al. (2001). stated that podocyte depletion leading to podocyte insufficiency and capillary collapse have been invoked as important steps in the development of FSGS. Apoptosis could constitute a vicious circle leading to more podocyte injury. On the other hand, apoptosis in DMP is not hard to explain given that the accumulation of ECM proteins by itself is an important incentive to apoptosis (Baker et al., 1994; Rost et al., 2002).

We have thus shown that apoptosis is increased in children with SRNS, particularly those with more severe pathology such as FSGS.

This study presented elevation of urinary annexin V in steroid-resistant nephrotic patients compared to controls. These results are in agreement with those reported by Simsek et al. (2008). Although, it was reported, based on limited data, that urinary annexin V concentration may be an indicator of renal injury related to the urinary protein level (Matsuda et al., 2000), other reports did not find a correlation between urinary annexin V and protein excretion (Simsek et al., 2008). In the current study, there was a significant elevation of MMP-9 in the study group, which was expected and matching with both experimental animal model and human studies (Krämer et al., 2008; Bauvois et al., 2007).

In children who did not respond to CPA, both urinary annexin V (p = 0.03) and the presence of apoptosis in renal biopsy specimens (p = 0.003, LH, 2.47) were significantly higher than responders. We could thus demonstrate the relation between increased apoptosis and CPA resistance. The difference in serum MMP-9 concentration between responders and non-responders did not reach significance (p = 0.08), while MMP-9 was higher (p = 0.05) in patients with biopsy evidence of apoptosis. Nevertheless, we c ould not demonstrate an association between urinary annexin V and apoptotic bodies in renal tissue.

Both annexin V and MMP-9 increased after treatment with CPA. Although, the increase in annexin V was less marked, it was still statistically significant. Despite that increase in both annexin V (Borzecka et al., 2004) and MMP-9 (Wasilewska and Zoch-Zwierz, 2008). Following specific therapy for nephrotic syndrome were previously reported, this would not be expected given that annexin V is an apoptotic marker and that MMP-9 was higher in those with tissue evidence of apoptosis.

Vu and Werb (2000) stated that MMP-9 can affect cell survival and proliferation both positively and negatively. Although, MMP-9 was reported to induce a pro-apoptotic signal similar to that of fibronectin (Schedin et al., 2000), MMP-9 activity may be required to generate a proliferative or anti-apoptotic signal (Uzui et al., 2000; Chang et al., 2006).

Especially that those with MCNS had higher MMP-9 (despite less apoptosis) compared to those with DMP, it is possible that MMP-9 may have counter-regulatory anti-apoptotic effects. To the same effect, Daniel et al. (2001) stated that tissue inhibitors of metalloproteinase (TIMPs) had an anti-inflammatory effect. The role of MMP-9 as a contributor to kidney disease progression and hence the potential use of TIMPs as an adjunct treatment of refractory cases of SRNS is the more important issue which needs further study.

In conclusion, apoptosis was demonstrated in most cases of FSGS presenting as SRNS as well as in some cases of DMP. Serum MMP-9 is elevated in SRNS and increases after treatment with cyclophosphamide. It may possess proliferative anti-apoptotic effect and may contribute to kidney disease progression. The potential therapeutic role of TIMPs needs further elucidation.