Apoptotic Markers in Childhood Nephrotic Syndrome
In order to investigate the status and role of serum MMP-9 and urinary annexin V in steroid resistant nephrotic syndrome, 60 children aged between 2 and 15 years were enrolled in this study. Serum MMP-9 and urinary annexin V excretion were measured N patients were examined for apoptotic bodies by DNA in situ. Serum MMP-9 was significantly higher in patients than controls (189.1± 150.6 and 87± 53.7 pg mL-1, respectively, p<0.0001). Urinary annexin V was also significantly higher (4.5± 0.3 compared to 2.7± 0.6 ng g-1 creatinine, p<0.0001). Apoptosis by DNA in situ was positive in 29 children (48%) and positive cases tend to have higher MMP-9 levels (p = 0.05). Those with focal segmental glomerulosclerosis had the highest apoptosis rates (69%) and the lowest response to CPA (29%). Responders had higher urinary annexin V (p = 0.03) and biopsy evidence of apoptosis (p = 0.003) than non-responders. Negative apoptosis by DNA in situ predicted response with a likelihood ratio of 2.47. These data confirm the role of annexin V and MMP-9 in apoptosis in nephrotic syndrome. The role of MMP-9 in disease progression and that of TIMPs as adjunctive therapy need further elucidation.
Apoptosis is a fundamental component of tissue differentiation and development
and plays an important role in a variety of diseases. Excessive apoptosis is
observed in diseases such as AIDS, Alzheimers and progressive heart failure.
On the other hand, insufficient apoptosis often occurs in tumor growth (Thatte
and Dahanukar, 1997) . Many renal diseases are characterized by mononuclear
cell infiltrate and inflammatory cells may provide factors that cause parenchymal
cell apoptosis (Tawfeek and Zaki, 2005). Nephrotic syndrome
is associated with and probably caused by, abnormalities in T lymphocyte function
(Zachwieja et al., 2003), which may be related
to increased apoptosis of circulating T lymphocytes (Zachwieja
et al., 2002).
Annexins are a superfamily of calcium- and phospholipids-binding proteins (Schiffer
et al., 2001) which originally evoked interest as mediators of the
anti-inflammatory actions of glucocorticoids (Ehrich et
al., 2007). Annexin V has been reported to possess anticoagulant activity,
inhibition of phospholipase A2 as well as regulation of membrane transport,
proliferation and signal transduction. Annexin V binds to the phosphatidyl serine-exposing
apoptotic cell and can inhibit the procoagulant and proinflammatory activities
of the dying cell (Schiffer et al., 2001). Children
with nephrotic syndrome were reported to have increased T lymphocyte apoptosis,
assessed by annexin V (Zachwieja et al., 2003;
Borzecka et al., 2004). Urinary annexin V excretion
may be an indicator of apoptosis and renal injury.
The progression of nephrotic syndrome to end stage kidney disease is accompanied
by accumulation of Extracellular Matrix Proteins (ECMs). Tissue matalloproteinases
(MMPs) are zinc-dependant endopeptidases known for their ability to cleave one
or several ECM constituents as well as non matrix proteins. They have a pivotal
role in control of signals elicited by matrix molecules (Vu
and Werb, 2000; Levick and Brower, 2008) and regulation
of cell migration and tissue morphogenesis (Herzog et
Based on the well-known ability of MMPs to degrade ECM components and their
ability to induce or sustain inflammatory mesangial cell phenotype (Bobkova
et al., 2008), MMPs are proposed to play an important role in the
progression of nephropathies (Ronco and Chatziantoniou,
2008). Moreover (Bauvois et al., 2007) suggested
that dysregulated renal expression of MMPs and their tissue inhibitors could
contribute to tubule-interstitial fibrosis characteristic of progressive primary
Matrix metalloproteinase 9, known as gelatinase B, is a member of the MMPs
which has been suggested to play a role in the degradation of ECM during inflammation,
wound healing and angiogenesis (Ohno et al., 1997).
It is also implicated in the processes of arthritis, cardiovascular diseases,
cancer metastasis and renal diseases (Massova et al.,
1998; Andreini et al., 2004).
The aim of this study was to define the status and role of MMP-9 and urinary annexin V excretion in children with Steroid-Resistant Nephrotic Syndrome (SRNS).
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:
||Initial clinical and basic laboratory evaluation
||Quantitation of annexin V in 24 h urine by enzyme-linked immunosorbent
assay (ELISA) for human annexin V using BMS252 kit (Bender Medsystems, Vienna,
Austria) (Borzecka et al., 2004)
||Measurement of serum total MMP-9 by ELISA using Quantikine
kit (R and D systems, USA) (Bauvois et al., 2007)
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).
||Detect apoptosis in renal tissue. 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)
||Treatment with oral cyclophosphamide (CPA), in a dose of 2.5
mg/kg/day, together with alternate day prednisone for three months according
to the standard protocol
||Assessment of the response to CPA. Responders were defined
as those who achieve clinical remission associated with normal protein excretion.
Partial responders were defined as having clinical remission and persistent
proteinuria below nephrotic range. Failure to achieve clinical remission
and/or persistent nephrotic range proteinuria defined resistance
||Repeat assessment of MMP-9 and urinary annexin V at the end
of CPA treatment
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).
|| 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
|| 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
||Mean serum matrix metalloproteinase-9 (MMP-9) concentrations
in control subjects, patients with steroid-resistant nephrotic syndrome
before and after treatment with cyclophosphamide
||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.
||Comparison of apoptotic markers between responders to CPA
and resistant cases
|Data expressed as Mean± SD, DNA in situ are
expressed as No. (%)
||MMP-9 and urinary annexin V according to DNA in situ
|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.
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