| Abstract|| |
Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are often studied together, because both present with heavy proteinuria and the nephrotic syndrome. The precise distinction between MCD and FSGS is sometimes difficult because of inadequate number of glomeruli for definite diagnosis. Some evidence suggests that markers of lipid peroxidation, such as Malondialdehyde (MDA) is an index of free radical mediated injury and may be involved in the pathogenesis of FSGS. In this study, we assessed the immunoreactivity of MDA, the end product of lipid peroxidation in glomeruli of patients with idiopathic FSGS, MCD as well as normal controls (NC). Our results showed that the immunostaining level of MDA was significantly higher in patients with FSGS (mean = 1.5) than in either patients with MCD (mean = 0.16) or normal controls (mean = 0.11) with P value < 0.001. Glomerular MDA level correlated well with the degree of glomerulosclerosis in patients with idiopathic FSGS. Our data demonstrates that the glomerular level of MDA is higher in idiopathic FSGS than MCD. We suggest that MDA immunostaining can be helpful in differentiating between FSGS and MCD in problematic cases and when we do not have enough glomeruli for definite and correct diagnosis.
|How to cite this article:|
Nezhad ST, Momeni B, Basiratnia M. Glomerular malondialdehyde levels in patients with focal and segmental glomerulosclerosis and minimal change disease. Saudi J Kidney Dis Transpl 2010;21:886-91
|How to cite this URL:|
Nezhad ST, Momeni B, Basiratnia M. Glomerular malondialdehyde levels in patients with focal and segmental glomerulosclerosis and minimal change disease. Saudi J Kidney Dis Transpl [serial online] 2010 [cited 2013 May 25];21:886-91. Available from: http://www.sjkdt.org/text.asp?2010/21/5/886/68886
| Introduction|| |
Minimal Change Disease (MCD) and idiopathic Focal and Segmental Glomerulosclerosis (FSGS) are relatively frequent glomerular diseases that cause heavy proteinuria and the nephrotic syndrome (NS). The characteristic ultrastructural morphological lesion in both of them is extensive flattening of podocyte foot processes, suggesting a primary defect in these cells.  FSGS shows segmental sclerosis and much more severe podocyte injury than MCD. 
The precise distinction between MCD and FSGS is sometimes difficult to make, especially in kidney biopsy samples with inadequate number of glomeruli. MCD is usually encountered in children and responds to treatment with steroids, whereas FSGS is more frequently diagnosed in adults and usually, does not respond to steroid therapy. FSGS shows a tendency to progress to end-stage renal disease (ESRD) and rapid recurrence after kidney transplantation. ,, Because of the focal nature of FSGS, the biopsy sample size becomes critical in diagnosing this glomerular disease. If the prevalence of segmentally scarred glomeruli is 10%, there is a 35% probability that no abnormal glomeruli will be found in the biopsy sample that contains only ten glomeruli resulting in misdiagnosis of MCD. The probability of missing the glomerular lesion drops to 12% or less when 20 or more glomeruli are present in the biopsy sample. , Furthermore, because the lesion initially affects the juxtamedullary glomeruli, it is important that a deep enough sample (corticomedullary junction) be obtained; hence, small biopsies not containing this portion are not suitable for correct diagnosis of the early stage of FSGS. , Additionally, FSGS has been divided into different histologic variants, whose prognosis is different from each other. 
Oxidative stress (imbalance of antioxidants and peroxidants, in favor of the latter) is considered to be a feature of some acute and most chronic renal diseases. ,,,, Malondialdehyde (MDA) is an important marker of peroxidation induced by oxidative stresses. There are a few studies on the urinary level of MDA in patients with renal disorders and only one study of MDA determination in glomeruli of patients with FSGS and MCD, to determine the pathogenesis of FSGS.  Also, the significance of determination of MDA level in the glomeruli of patients with different subtypes of FSGS and MCD, for differentiation between these two diseases in renal biopsies, has not been evaluated.
| Materials and Methods|| |
A total of 91 patients with MCD and FSGS were originally collected for this study. All light microscopic slides were fixed immediately in 10% neutral formaldehyde overnight, dehydrated with alcohol and embedded in paraffin. Subsequently, 3 ΅m slides were prepared and stained as follows: two H & E, one PAS, one Masson trichrome and one silver stain. Cases of FSGS were sub-classified according to the proposal working classification.  Patients less than two years and more than 50 years old were excluded from the study.
Finally, 72 cases with adequate number of glomeruli were selected including 34 patients with idiopathic FSGS, 31 patients with MCD, three cases as normal controls which were selected from non-tumoral part of nephrectomy specimens and four patients with borderline pathology (these cases did not respond to steroid therapy but pathologic findings did not fulfill diagnosis of FSGS).
For cases of idiopathic FSGS, clinical and histologic data including sex, age, number of glomeruli, number of glomeruli with global and segmental sclerosis, presence or absence of mesangial cell proliferation, visceral epithelial hyperplasia, foam cells, hyalinosis, synechia, tubular atrophy, interstitial inflammation and interstitial fibrosis, were gathered and FSGS subtypes were determined. For cases of MCD, criteria such as sex, age, number of glomeruli and presence or absence of mesangial cell proliferation were gathered.
For immunohistochemical examination of glomerular malondialdehyde (MDA), avidin-biotinperoxidase complex staining method was employed. The tissue sections were first deparaffinated in xylene and then subsequently washed with 100%, 95% and 70% ethanol and rinsed with distilled water. Endogenous peroxidase activity was blocked with 0.3% H2O2, followed by washing with phosphate buffered saline (PBS). The tissues were then blocked with 5% goat serum. A primary antibody, specific for MDA (polyclonal antibody from rabbit Alexis ALX-210-879) (Calbiochem. Co.), was used for immunostaining. The tissues were washed with PBS and incubated by biotinylconjugated secondary antibody (K0673, Dako) with final washing with PBS following which hematoxylin staining was done. Sections were observed with an optical photomicroscope (Olympus, Japan).
Glomerular staining was graded in each glomeruli semiquantitiavly and separately scored from 0-4 (+) according to staining intensity. We evaluated all glomeruli in each case to obtain the final score and then determined the median score for each case. Mean score for each group and also in different subtypes of FSGS were also calculated.
| Statistics|| |
All data were expressed as mean and median and t-test was used to compare the differences between each group.
| Results|| |
The 34 patients with idiopathic FSGS included 31 males and three females with a mean age of 34 ± 8 years. According to the proposal working classification,  15 cases were NOS variant, seven were perihilar, eight were cellular, three were tip lesions and one case was of the collapsing variant [Table 1].
The 31 patients with MCD included 22 females and nine males with mean age of 21 ± 7 years. Twenty cases of MCD had mild mesangial cell proliferation.
The four patients with nephrotic range proteinuria, whose pathologic findings on needle biopsies were in favor of MCD, but did not respond to steroid therapy, were classified as borderline cases, and included three females and one male. Three of them showed mild mesangial cell proliferation.
The median immunostaining score for glomerular MDA was significantly higher in patients with FSGS than MCD and normal controls [Table 2]. Also, the mean of immunostaining of glomerular MDA was significantly higher in patients with FSGS (mean, 1.5) than in either the patients with MCD (mean, 0.16) or normal controls (mean 0.11) with P value < 0.001 [Table 3] and [Figure 1].
|Table 2 :Median score of malondialdehyde staining in different patients.|
Click here to view
|Figure 1 :Representative figures show immunoreactivity of Malondialdehyde (MDA) in the glomeruli of|
(A) a control kidney, (B) patient with minimal change disease and (C) patient with focal and segmental
glomerulosclerosis. Glomerular staining is score zero in normal control, score 1 in minimal change disease
and increased (score 4) in the patient with focal and segmental glomerulosclerosis.
Click here to view
The glomerular MDA levels correlated well with the degree of glomerulosclerosis in patients with idiopathic FSGS. The results in borderline cases were similar to that in cases with FSGS (mean, 1.25).
| Discussion|| |
Our study demonstrated that glomerular levels of MDA are increased in patients with idiopathic FSGS. Minimal change disease and FSGS both typically present with the NS and cannot be readily distinguished based solely on clinical presentation. In children, the NS is presumed to be due to MCD and biopsy is only performed if the child is steroid unresponsive or has clinical features suggesting another etiology of the NS. 
The ultimate prognosis between MCD and FSGS and also between FSGS variants according to the proposal working classification differs dramatically, with complete recovery as seen in MCD, contrasting with progressive renal insufficiency in FSGS. , Sometimes, the distinction between MCD and FSGS is difficult, especially with small biopsy guns and needles. At least 23 glomeruli are needed to diagnose segmental lesions such as FSGS.  A sample with only ten glomeruli has a 35% probability of missing FSGS. 
Some studies have demonstrated that histological criteria such as glomerular enlargement, glomerular tip lesion, visceral epithelial cell hypertrophy, extracapilllary proliferation, glomerular hyalinosis, synechia, interstitial fibrosis and presence of patchy tubular atrophy are in favor of FSGS, but no one can be sure to diagnose FSGS without demonstrating segmental collapse of capillary lumens in needle biopsy. , Because of this limitation in detection of segmental lesions, other diagnostic features in glomeruli are needed for diagnosing FSGS, even in non-sclerotic glomeruli.
MDA is an important and common marker of lipid peroxidation induced by oxidative stress. , It reflects the interaction between mo-lecular oxygen and polyunsaturated fatty acids. Biologic membranes have high polyunsaturated fatty acid content, thus making them particu-arly susceptible to peroxidase attack by reac-tive oxygen species (ROS). , Lipid peroxi-dation can alter membrane structure and func-tion. , Because of the difficulties in measu-ring oxygen free radicals directly, lipid peroxi-dation is often used as an index of free radical mediated injury.
In one animal study, Gwinner et al, demonstrated that the superoxide anion production was ten-folds higher in glomeruli than tubules, while the glomerular activities of antioxidant enzymes were only one-third to one-sixth that of the proximal tubules, indicating that glomeruli are vulnerable to oxidative injury in vivo. 
Binder et al, demonstrated that glomerular production of oxygen free radicals was increased in Mpv 17 gene inactivated mice, a model of steroid resistant nephrosis similar to human FSGS. 
Budisavljevic et al showed that glomerular production of ROS increased 400-500% in rats with anti-thy 1 nephritis.  Ming Chang et al, also demonstrated a significant elevation of plasma, urinary and glomerular MDA levels in rats and patients with diabetic nephropathy and concluded that there is close relationship between oxidative stress and diabetic nephropathy. 
Ong Ajyooth et al, and Ece et al, in two different studies showed that oxidative stress may play an important role in the pathogenesis of IgA nephropathy and Henoch-Schonlein purpura. , Lee et al, claimed that lipid peroxidation may be involved in the pathogenesis of FSGS and dietary antioxidants may inhibit lipoprotein oxidation and renal injury in patients with FSGS.  Thus, all the above studies have shown that oxidative stress may play an important role in the pathogenesis of some glomerular diseases.
There is one study by Kuo et al, in which they have shown that urinary and plasma MDA levels significantly increase in patients with FSGS compared with MCD. They also demonstrated that immunostaining for glomerular MDA and SOD (superoxide dismutase) is higher in rats and humans with FSGS than those with MCD. 
In this study, we assessed immunoreactivity of glomerular MDA in patients with idiopathic FSGS and MCD to determine if it is possible to differentiate between these two common causes of the NS in problematic cases. We demonstrated that there is significant difference between the median score of these two conditions. The immnuostaining for glomerular MDA was significantly higher in patients with FSGS (mean, 1.5) than in either patients with MCD (mean, 0.16) or normal controls (P value < 0.01). There was no significant difference in MDA immunostaining levels between different variants of FSGS.
In all four borderline cases who presented with the NS not responding to steroid therapy and in whom, we did not find glomerular sclerosis in the kidney biopsies, glomerular MDA level were higher than MCD patients and was similar to the level in patients with FSGS (mean, 1.25).
In summary, our study demonstrates that glomerular levels of MDA are increased in patients with idiopathic FSGS, when compared with MCD. We recommend that MDA immunostaining of kidney biopsy samples with in adequate number of glomeruli can be helpful in differentiating between FSGS and MCD in problematic cases.
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Simin Torabi Nezhad
Professor of Pathology, Shiraz Nephrology Center, Pathology Department, Shiraz University of Medical Sciences, Shiraz
[Table 1], [Table 2], [Table 3]