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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 21  |  Issue : 5  |  Page : 886-891
Glomerular malondialdehyde levels in patients with focal and segmental glomerulosclerosis and minimal change disease


Shiraz Nephrology Center, Shiraz University of Medical Sciences, Shiraz, Iran

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Date of Web Publication31-Aug-2010
 

   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 2014 Apr 23];21:886-91. Available from: http://www.sjkdt.org/text.asp?2010/21/5/886/68886

   Introduction Top


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. [1] FSGS shows segmental sclerosis and much more severe podocyte injury than MCD. [2]

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. [1],[3],[4] 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. [1],[5] 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. [1],[5] Additionally, FSGS has been divided into dif­ferent histologic variants, whose prognosis is different from each other. [6]

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. [7],[8],[9],[10],[11] 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. [12] 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 Top


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. [6] 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 Top


All data were expressed as mean and median and t-test was used to compare the differences between each group.


   Results Top


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, [6] 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].
Table 1 :Clinical and histologic characteristics of patients with FSGS.

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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.

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Table 3 :Mean score of malondialdehyde staining in different patients.

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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.


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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 Top


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. [1]

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. [1],[6] 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. [5] A sample with only ten glomeruli has a 35% pro­bability of missing FSGS. [1]

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. [1],[13] 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. [14],[15] 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). [15],[16] Lipid peroxi-dation can alter membrane structure and func-tion. [12],[17] 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. [18]

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. [19]

Budisavljevic et al showed that glomerular production of ROS increased 400-500% in rats with anti-thy 1 nephritis. [20] 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. [16]

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. [10],[11] 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. [21] 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. [12]

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.

 
   References Top

1.Jennette JC, Olson JL. The nephrotic syn­drome. Heptein Stall,S Pathology of the Kid­ney, 6th edition. Boston, Little brown; 2007: 125-205.  Back to cited text no. 1      
2.Shi SF, Wang SX, Zhang YK, Zhao MH, Zou WZ. Ultra structural features and expression of Cyclosketon proteins of podocyte from patients with minimal change disease and focal seg­mental Glomerulosclerosis. Ren Fail 2008;30 (5):477-83.  Back to cited text no. 2      
3.Gultai S, Sengupta D, Sharma RK, et al. Steroid resistant nephrotic syndrome: Role of histopathology. Indian Pediatr 2006;43(1):55­60.  Back to cited text no. 3      
4.Savin VS, Sharma RK, Sharma A, Mecarthy ET, Swan SK, Ellis E. Circulating factor asso­ciated with increased glomerular permeability to albumin in recurrent focal segmental Glo­merulosclerosis. N Engl J Med 1996;334:878­-83.  Back to cited text no. 4      
5.Fogo AB. Approach to renal biopsy. Am J Kidney Dis 2003 ;42(4):826-36.  Back to cited text no. 5      
6.Vivette DD, Fogo AB, Bruijin JA, Jennette JC. Pathologic classification of focal segmental Glomerulosclerosis. A working proposal clas­sification. Am J Kidney Dis 2004;43(2):368­-82.  Back to cited text no. 6      
7.Agarwal R. Chronic kidney disease is associa­ted with oxidative stress independent of hyper-tension. Clin Nephrol 2004;61:377-83.  Back to cited text no. 7  [PUBMED]    
8.Atanasiu V, Stoian I, Capusa C, et al. Oxi­dative stress parameters in hemodialysis pa­tients with or without diabetes. Rom J Intern Med 2006;44(4):433-42.  Back to cited text no. 8      
9.Kocak H, Gumuslu S, Ermis C, et al. Oxidative stress and asymertric dimethylarginne is inde­pendently associated with carotid intima media thickness in peritoneal dialysis patients. Am J Nephrol 2008;28(1):91-6.  Back to cited text no. 9      
10.Ong-Ajyooth L, Ong-Ajyooth S, Parichatikanond P. The effect of a - tocopherol on the oxidative stress and antioxidants in idiopathic Ig A neph­ropathy. J Med Assco Thai 2006 ;89(s5):5164-­70.  Back to cited text no. 10      
11.Ece A, Kelekci S, Kocamaz H, et al. Anti­oxidant enyzyme activities, lipid peroxidation, and total antioxidant status in children with Henoch - Schonlein purpura. Clin Rheumatol 2006;27(2):163-9.  Back to cited text no. 11      
12.Kuo HT, Kuo MC, Chiu YW, Chang JM, Guh JY, Chen HC. Increased glomerular and extra­cellular Malondialdehyde levels in patients and rats with Focal Segmental Glomerulosclerosis. Eur J Clin Invest 2005;35:245-25.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]  
13.Howie AJ. Pathology of minimal change neph­ropathy and segmental sclerosing glomerular disorders. Nephrol Dial Transplant 2003;18 (suppl 6):vi33-8.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Freeman BA, Crapo JD. Free radicals and tissue injury. Lab Invest 1982;47:412-6.  Back to cited text no. 14  [PUBMED]    
15.Chance B, Sies H, Boveris A. Hydroxide meta­bolism in mammaline organs. Physiol Rev 1979;59:527-605.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]  
16.Ming Chang J, Chuan Kuo M, Kuo HT, Wen Chiu YI, Chun Chen H. Increased glomerular and extracellular Malondialdehyde levels in patients and rats with diabetic nephropathy. J Lab Clin Med 2005;146(4):210-5.  Back to cited text no. 16      
17.Slater TF. Free radical mechanisms in tissue injury. Biochem J 1984;222:1-15.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]  
18.Gwinner W, Deters-Evers U, Brandes RP, Kubat B, Koch KM, Pape M. Antioxidant balance in the glomerulus and proximal tubules of rat kidney. J Physiol 1998;509:599-606.  Back to cited text no. 18      
19.Binder CJ, Weiher H, Exner M, Kerjaschki D. Glomerular overproduction of oxygen free ra­dicals in Mpv 17 gene inactivated mice caused podocytes foot process flattening and pro­teinuria. A model of steroid resistant nephrosis sensitive to radical scavenger therapy. Am J Pathol 1999;154:1067-75.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]  
20.Budisavljevic MN, Hodge L, Barber K, Fulmer JR, Durazo-Arvizu RA, Self SE. Oxidative stress in pathogenesis of experimental mesan­giproliferative glomerulonephritis. Am J Physiol Renal Physiol 2003;285:1138-48.  Back to cited text no. 20      
21.Lee HS, Jeong JY, Kim BC, Kim YS, Zhang YZ, Chung HK. Dietary antioxidant inhibits lipoprotein oxidation and renal injury in expe­rimental FSGS. Kidney Int 1997;51(4):1151-9.  Back to cited text no. 21      

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Correspondence Address:
Simin Torabi Nezhad
Professor of Pathology, Shiraz Nephrology Center, Pathology Department, Shiraz University of Medical Sciences, Shiraz
Iran
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