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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2018  |  Volume : 29  |  Issue : 4  |  Page : 893-901
Study of glucocorticoid receptors in T lymphocytes (CD3/GCR) as predictor of steroid responsiveness in Egyptian children with idiopathic nephrotic syndrome


1 Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Sharqia, Egypt
2 Department of Pediatrics, Faculty of Medicine, Tanta University, Tanta, Gharbia, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Sharqia, Egypt

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Date of Submission21-Sep-2017
Date of Acceptance29-Nov-2017
Date of Web Publication28-Aug-2018
 

   Abstract 

Little was known about the relationships between the T lymphocytes (CD3+), expression of glucocorticoid receptors (GCR) and the response to GC treatment in children with the idiopathic nephrotic syndrome (INS). Our objective was to determine the relation between steroid responsiveness and GCR expression in T lymphocytes. The present study was carried out on 80 children with new-onset INS admitted in Pediatric Nephrology Units of Zagazig and Tanta University Hospitals and on 40 healthy children of the same age and sex who served as control group. The Subjects were subdivided into three groups as follows: Group 1 with 40 healthy children of comparable age and sex served as control group; Group 2 consisted of 60 patients diagnosed with INS with early response to steroid therapy [early responder (ER)] and Group 3 with 20 patients diagnosed with INS with late response to steroid therapy [late responder (LR)]. They were subjected to history taking, focusing on the pattern of response to steroids (ERs), clinical examination, routine laboratory investigations and the GCR/CD3% relationship. 75% of newly diagnosed INS cases were ER whereas 25% were LR. GCR/CD3% was significantly lower in LR group in comparison with ER and control groups, with a significant negative correlation between time of steroid responsiveness and GCR/CD3%. LR group showed lower GCR expression in T lymphocytes before starting therapy which may mean that GCR expression could be part of a pathophysiological mechanism of steroid responsiveness in these children and can be used as a useful diagnostic marker to predict steroid responsiveness in patients with INS.

How to cite this article:
Amin EK, El-Gamasy MA, Shokry DM, Khalifa NA. Study of glucocorticoid receptors in T lymphocytes (CD3/GCR) as predictor of steroid responsiveness in Egyptian children with idiopathic nephrotic syndrome. Saudi J Kidney Dis Transpl 2018;29:893-901

How to cite this URL:
Amin EK, El-Gamasy MA, Shokry DM, Khalifa NA. Study of glucocorticoid receptors in T lymphocytes (CD3/GCR) as predictor of steroid responsiveness in Egyptian children with idiopathic nephrotic syndrome. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2020 Jul 13];29:893-901. Available from: http://www.sjkdt.org/text.asp?2018/29/4/893/239659

   Introduction Top


Glucocorticoids (GCs) are the treatment of choice for young patients with the idiopathic nephrotic syndrome (INS). However, some patients fail to respond to the treatment even when given high dose GC. For those patients, the treatment should be bolstered by synergizing with other immunosuppressants.[1]

Although biochemical alterations and clinical manifestations in most nephrotic patients seem to be quiet similar, substantial differences are encountered regarding the course of the disease. Relapse of proteinuria is experienced in approximately 60%–80% of steroid-sensitive patients, and despite initial complete remission, some remain steroid-dependent or become steroid-resistant.[2]

The lack of response to corticosteroids has been explained by several mechanisms. It may be ascribed to overwhelming disease severity, poor compliance, abnormalities in glucocor-ticoid metabolism, poor absorption and/or GC resistance due to GC receptor (GCR) abnormality. GCR was incriminated in worsening the response to steroids either due to inherited target tissue defective response or acquired impaired responsiveness.[2],[3]


   Subjects and Methods Top


Study design

The present case-control study was carried out after approval from the Research Ethical Committee centers of Zagazig and Tanta University Hospitals and obtaining informed consents from parents of included children. Eighty patients with new-onset INS admitted in Pediatric nephrology units of Zagazig and Tanta University Hospitals who were categorized prospectively according to their response to GCs were included in the study and 40 healthy children of the same age and sex served as control group. The subjects were subdivided into three groups; Group 1 with 40 healthy children of comparable age and sex served as control group (their ages ranged from one to eight years with a mean of 4.3 ± 1.9 years, they were 20 males and 20 females); Group 2 consisted of 60 patients diagnosed as INS with early response to steroid therapy [early responder (ER)] who achieved complete remission within four weeks of starting steroid therapy (their age ranged from 2–5 years with a mean of 3.2 ± 1.3 years, they were 42 males and 18 females) and Group 3 with 20 patients diagnosed as INS with late response to steroid therapy [late responder, (LR)] who achieved complete remission later on from four to six weeks of starting steroid therapy (their age ranged from 2 to 6 years with a mean of 3.9 ± 1.4 years, they 8 males and 12 females).

Inclusion criteria

All children admitted to the two university hospitals with new-onset INS aged from one to eight years old during the period from March 2016 to March 2017.

Exclusion criteria

Atypical nephrotic syndrome suspected by the age before one year of life, hypertension, macroscopic hematuria, abnormal urinary sediments (abnormal casts or crystaluria) or elevated serum creatinine, known cases of the secondary nephrotic syndrome, patients with a history of steroid intake for any cause for the six months before the study.

INS was diagnosed according to Kidney Disease Improving Global Outcome guidelines by the presence of nephrotic range proteinuria >40 mg/m2/h, generalized edema, hyperlipidemia and hypoalbuminemia <2.5 g/dL. Steroid sensitive nephrotic syndrome (SSNS) was diagnosed if the patients respond to the daily steroid full dose therapy (60 mg/m2/day) within six weeks. A complete remission was defined by marked reduction in proteinuria (to <4 mg/m2/h, or urine albumin dipstick of 0 to trace for three consecutive days) in association with resolution of edema. Steroid dependency was defined as an initial remission followed by two or more relapses during the steroid reduction period or within 15 days after tapering of prednisone. A relapse was defined by the recurrence of severe proteinuria (>40 mg/m2/h, or urine albumin dipstick >3+ on 3 successive days), often with a recurrence of edema. Steroid-resistant nephrotic syndrome (SRNS) was considered after six weeks of daily full dose steroid plus three methylprednisone pulse therapy without improvement of proteinuria.[4]

Protocol of treatment

All patients were at the 1st episode of nephrotic syndrome and initially were treated by prednisone (60 mg/m2/day) for a full duration of 30 days. While relapses were treated by oral prednisone at 60 mg/m2/day until remission plus five additional days, then the prednisone dose was tapered to 40 mg/m2 every other day (EOD) for four weeks, followed by a reduction of 15 mg/m2 EOD per week to the lowest dose necessary to maintain remission and, ultimately, discontinuation of treatment if appropriate.[5] All patients and controls were subjected to the following:

  1. History taking: about age, sex, the onset of symptoms, drug intake, comorbidities, response to steroid therapy, steroid-related adverse effects (as obesity, hypertension, bone ache, and short stature), other immu-nosuppressive drugs adverse effects
  2. Clinical examination: general examination (blood pressure, weight, edema), percussion for ascites, organomegaly, percussion for pleural effusion or chest infection, and percussion for pericardial effusion
  3. Laboratory investigations: which included complete urine analysis, 24-h urinary collection for volume, urinary protein/ creatinine ratio, complete blood counting (CBC), blood urea nitrogen (BUN) and serum creatinine serum albumin, serum cholesterol) by automated autoanalysis (OBAS500) and the percentage of lymphocytes containing glucocorticod receptors (CD3/GCR).[6]


Sampling for laboratory investigations

About 6 mL venous blood sample was withdrawn from patients and controls, 2 mL blood sample was used for CBC estimation using EDTA vacutainer tubes and the remainder of blood were put into plain tubes for centri-fugation and separation of serum which used for estimation of cholesterol, urea, creatinine and albumin levels.

The urine samples were collected from patients in the early morning during the first week of the onset of nephrotic syndrome and also from controls by one of the two techniques, either a clean catch mid stream method or urinary Catheterization method using 8 French polyethylene feeding tube.

The urine samples were put into sterile containers then centrifuged for 20 min.

The urinary collection samples were kept into sterile containers refrigerated at 2°C–8°C and then the samples were re-warmed to room temperature just before laboratory assessment.[7]

Flow cytometry analysis of the percentage of lymphocytes (CD3/glucocorticoid receptor)

Identification of GCR/CD3 is obtained by flow cytometer immunophenotyping with purified monoclonal anti-GCR labeled antibodies conjugated with fluorescein isothiocyanate and anti-CD3 antibodies conjugated with phyco-erythrin.[6],[8]


   Statistical Analysis Top


Data were checked, entered and analyzed by using Statistical Package for Social Sciences (SPSS) version 20.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were expressed as (mean ± standard deviation) for quantitative variables, number and percentage for categorical variables, Chi-squared (χ2) test was used to find the relationship between qualitative data, t-test, ANOVA (F-test), correlation coefficient (r) were used when appropriate to explore the relationship between quantitative data, P>0.05 was considered statistically significant and P>0.001 was considered highly significant.[9]


   Results Top


[Table 1] summarizes the demographic and clinical data of the studied patients and controls. The ages of the first group are ranging between one to eight years with mean (4.3 ± 1.9), The ages of the second group are ranging between two to five years with mean (3.2 ± 1.3) and The ages of the third group are ranging between two to six years with mean (3.9 ± 1.4) with no statistically significant difference between the three groups As regarding sex, there is no statistically significant difference between the three groups (P>0.05).
Table 1: Demographic and clinical data of the studied patients and control.

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[Table 2] summarizes laboratory data of the studied patients and controls. There is no statistically significant difference between the ER and LR groups as regards urinary protein/ urine creatinine ratio, serum albumin, cholesterol, Hb, BUN, and serum creatinine. There is significant increase in upr/creatinine ratio and serum cholesterol levels in the diseased (ER and LR) groups when compared to controls. There was a significant decrease in serum albumin in the diseased (ER and LR) groups when compared to controls.
Table 2: Laboratory data in studied patients and control.

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As regards GCR, there is highly statistically significant difference between the early and late response groups. [Figure 1]a, [Figure 1]b and [Figure 2]a, [Figure 2]b (P = 0.000), the percentage was nearly equal in both ER and control groups and is significantly decreased in LR group.
Figure 1a and b: The percentage of lymphocytes containing glucocorticoid receptors (CD3/GCR) by flow cytometry in some of early responder group.

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Figure 2a and b: The percentage of lymphocytes containing glucocorticoid receptors (CD3/GCR) by flow cytometry in some of late responder group.

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[Table 3] shows correlations between the time of response to steroid therapy and other studied demographic and laboratory variables. There is no statistically significant correlation between the time of response to steroid therapy and other studied demographic and laboratory variables (P >0.05).
Table 3: Correlation between the time of response to steroid therapy and studied demographic and laboratory markers.

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[Figure 3] shows that there is no statistically a significant correlation between time of response to steroid therapy and urinary protein to urinary creatinine ratio (P>0.05).
Figure 3: Correlation between the time of response in weeks and u protein/creatinine.

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[Table 4] summarizes correlations between GCR% and other studied laboratory variables, there was highly statistically significant negative correlation between GCR and time to the response which means the more the percentage of GCR, the lesser the time needed to induce complete remission (P = 0.000) [Figure 4] but there is no statistically significant correlation between time of GCR% and other studied laboratory variables (P>0.05).
Table 4: Correlation between GCR and laboratory markers.

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Figure 4: Correlation between the time of response and glucocorticoid receptors.

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   Discussion Top


One of the most important indicators for the outcome of children with INS is their initial response to GC treatment.[10] Being able to predict the clinical response before starting steroid therapy can help to decide about the need of administration of synergized therapy for the treatment at the beginning itself and this will help avoid the side effects of chronic highdose steroid therapy, improve the individual response to GC therapy and benefit more patients.[4],[11]

We hypothesized that evaluation of GCR expression in children by a suitable method before treatment might be beneficial in predicting response to steroids. In our study, of the total 80 patients, ER to steroid therapy was 60 (75%), and only 20 (25%) were LR. This prevalence of ER over LR was in agreement with Kaddah et al, 2012 who aimed to define the characteristics of the primary nephrotic syndrome in Egyptian children. In their study, 66% of studied children were ER and 44% were LR.[12]

This study included nephrotic patients with age group ranged from one to six years, and this was the most common age-group at the presentation of INS in pediatric age. This finding was in keeping with previously reported work of Araya et al.[13] Our study also showed a predominance of male patients over females which was similar to previous studies elsewhere including Sinha et al.[14]

In our study, there were no statistically significant differences between the time of steroid responsiveness and the age of the studied patients. This finding was in contrast to an earlier report which documented intense relationship between the age and steroid response, but this study was done on a wider range age groups comparing steroid sensitive with steroid-resistant cases, but in our work, we have included only a narrow age group of nephrotic patients to be sure that our cases are all idiopathic and mostly diagnosed as minimal change disease.[15]

In our study, there was no statistically significant differences between ER and LR as regarding gender distribution which was in agreement with the study of Baker, who reported no statistically significant association between steroid response and gender distri-bution.[16] In our study, there was no statistically significant differences between ER and LR groups as regarding routine laboratory findings including urinary protein to urinary creatinine ratio or serum creatinine. This is in agreement with Mortazavi and Khiavi who evaluated the steroid response pattern and outcome of INS and found no statistically significant differences in laboratory findings between their studied patients.[17]

This finding was also supported by Alchi and Jayne, who studied urinary protein markers as predictors to the severity of renal histological lesions in children with mesangial proliferative glomerulonephritis and found no significant differences in laboratory findings between their studied patients.[18]

Furthermore, we analyzed the correlations between the time interval from the start of GC therapy to complete remission and the percentage of T lymphocytes (CD3+) expression of GCR. The mean percentage of lymphocyte expression of GCR (CD3+/GCR) was significantly lower in the LR group than that in the ER group and healthy group. The CD3/GCR% was significantly higher in the patients who achieved complete remission within the 1st week of steroid treatment compared to patients who did not achieve complete remission until the 6th week after initiation of steroid therapy.[7]

This is in agreement with Wasilewska et al, who studied the expression of GCR in mono-nuclear cells in 23 children with SSNS by flow cytometry. T lymphocyte (CD3+) expression of GR was significantly lower in LR than in the control group, whereas it was similar in ER and control groups.[19] Furthermore, Szilagyi et al, observed lower expression of GCR tested by the Western blotting in SRNS than in SSNS and controls.[20]

Another study done by Bagdasarova et al, assessing GCR expression in monocytes by immunohistochemistry reported a lower level of GR in SRNS than in SSNS patients.[21]

Our work was in accordance with a study done by Han et al, on adult patients diagnosed by renal biopsy to have minimal change nephrotic syndrome using immunohistoche-mistry to demonstrate GCR-mRNA on the glomeruli. This study concluded that glome-rular GCR expression is decreased in patients whose response to GCs is delayed. In addition, this study shows that the time interval from the start of steroid treatment to complete remission was inversely correlated with the mRNA expression of GCR in glomeruli.[22]

On the contrary, Haack et al investigated GCR in 28 pediatric patients with INS by testing the density and binding affinity of the receptors using a dexamethasone binding assay. They found no significant differences between SSNS and SRNS patients and between them and healthy Controls. The variations in these studies may be due to the timing of sampling, use of heterogeneous material or different analytical techniques for the assessment of GR.[23]

An important finding in our study is that the expression of intracellular GCR shown as the percentage of lymphocytes containing the receptors was found to be negatively correlated to the time interval from the start of steroid treatment to complete remission. In Arab world 2016, a similar study had been done on 60 newly diagnosed nephrotic cases, and a similar number representing control group showed a significant inverse correlation between the expression of intracellular GCR (CD3/GCR) and the time interval from the start of steroid therapy to complete remission.[24]

The study had identified the percentage of GCR/CD3 as a predictor of the future course of INS as regarding the future responsiveness or relapses. This was justified by Vivarelli et al, who concluded that the length of time between steroid treatment onset and remission is an early prognostic indicator for patients with INS.[25] Furthermore, Zahran et al, had concluded that evaluation of the expression of intracellular GCR in T lymphocytes at time of diagnosis of INS could predict the response to steroid therapy and can help in the determination of the outcome of NS patients regarding future relapses.[26]


   Conclusions Top


We conclude that the prescence of abundant GCR in ER rather than in LR groups shows that GCR% can be used as a useful diagnostic marker to predict steroid responsiveness, resistance or dependence in children with INS.


   Recommendations Top


We recommended evaluation of the expression of intracellular GCR % of lymphocytes which can possibly directly predict early and late responders to steroid therapy, and as a result, the outcome of nephrotic patients could be predicted.

Conflict of interest: None declared.

 
   References Top

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Liu D, Ahmet A, Ward L, et al. A practical guide to the monitoring and management of the complications of systemic corticosteroid therapy. Allergy Asthma Clin Immunol 2013; 9:30.  Back to cited text no. 1
    
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Liang Y, Chen Y, Chen Y, Gong Y. Role of the glucocorticoid receptor in the recurrence of primary nephrotic syndrome. Exp Ther Med 2015;10:1556-62.  Back to cited text no. 2
    
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Che R, Zhang A. Mechanisms of glucocorticoid resistance in idiopathic nephrotic syn-drome. Kidney Blood Press Res 2013;37:360-78.  Back to cited text no. 3
    
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Lombel RM, Gipson DS, Hodson EM; Kidney Disease: Improving Global Outcomes. Treatment of steroid-sensitive nephrotic syndrome: New guidelines from KDIGO. Pediatr Nephrol 2013;28:415-26.  Back to cited text no. 4
    
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Ravani P, Magnasco A, Edefonti A, et al. Short-term effects of rituximab in children with steroid-and calcineurin-dependent nephrotic syndrome: A randomized controlled trial. Clin J Am Soc Nephrol 2011;6:1308-15.  Back to cited text no. 5
    
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Zare H, Shooshtari P, Gupta A, Brinkman RR. Data reduction for spectral clustering to analyze high throughput flow cytometry data. BMC Bioinformatics 2010;11:403.  Back to cited text no. 6
    
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Vaidya VS, Waikar SS, Ferguson MA, et al. Urinary biomarkers for sensitive and specific detection of acute kidney injury in humans. Clin Transl Sci 2008;1:200-8.  Back to cited text no. 7
    
8.
Murphy RW, Lowcock LA, Smith C, Darevsky IS, Orlov N. Flow cytometry in biodiversity surveys: methods, utility and constraints. Amphibia Reptilia 2007;27(1):1813.  Back to cited text no. 8
    
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Khothari CR, editors. Research Methodology, Methods and Techniques, New Age International. 2nd ed. New Delhi 2012. p. 95-7.  Back to cited text no. 9
    
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Sinha A, Saha A, Kumar M. Extending initial prednisolone treatment in a randomized control trial from 3 to 6 months did not significantly influence the course of illness in children with steroid-sensitive nephrotic syndrome. Kidney Int 2015; 87(1):217-24.  Back to cited text no. 10
    
11.
Hodson EM, Willis NS, Craig JC. Non-corticosteroid treatment for nephrotic syndrome in children. Cochrane Database Syst Rev 2008;1:CD002290.  Back to cited text no. 11
    
12.
Kaddah A, Sabry S, Emil E, El-Refaey M. Epidemiology of primary nephrotic syndrome in Egyptian children. J Nephrol 2012;25:732-7.  Back to cited text no. 12
    
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Araya CE, Wasserfall CH, Brusko TM, et al. A case of unfulfilled expectations. Cytokines in idiopathic minimal lesion nephrotic syndrome. Pediatr Nephrol 2006;21:603-10.  Back to cited text no. 13
    
14.
Sinha A, Hari P, Sharma PK, et al. Disease course in steroid sensitive nephrotic syndrome. Indian Pediatr 2012;49:881-7.  Back to cited text no. 14
    
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Anderson RF, Thrane N, Noergaard K, Rytter L, Jespersen B, Rittig S. Early age at debut is a predictor of steroid-dependent and frequent relapsing nephrotic syndrome. Pediatr Nephrol 2010;25(7):1299-304.  Back to cited text no. 15
    
16.
Baker DE. Demographic characters in NS. Nephrol Prog 2010;57:150-6.  Back to cited text no. 16
    
17.
Mortazavi F, Khiavi YS. Steroid response pattern and outcome of pediatric idiopathic nephrotic syndrome: A single-center experience in Northwest Iran. Ther Clin Risk Manag 2011;7:167-71.  Back to cited text no. 17
    
18.
Alchi B, Jayne D. Membranoproliferative glomerulonephritis. Pediatr Nephrol 2010;25: 1409-18.  Back to cited text no. 18
    
19.
Wasilewska A, Zoch-Zwierz W, Tomaszewska B, Wiercinski R, Stasiak-Barmuta A. Expression of glucocorticoid receptors in mononuclear cells in nephrotic syndrome. Pediatr Nephrol 2003;18:778-82.  Back to cited text no. 19
    
20.
Szilagyi K, Podracka L, Franke NE, Mojzis J, Mirossay L. A new link between steroid resistance, glucocorticoid receptor and nuclear factor kappa B p65 in idiopathic nephrotic syndrome. Neuro Endocrinol Lett 2009;30: 629-36.  Back to cited text no. 20
    
21.
Bagdasarova IV, Ivanov DD, Afanaseva VV. The morpho-functional characteristics of the blood lymphocytes in steroid-sensitive and steroid-resistant glomerulonephritis. Pediatr Nephrol 2013;102:7.  Back to cited text no. 21
    
22.
Han SH, Park SY, Li JJ, et al. Glomerular glucocorticoid receptor expression is reduced in late responders to steroids in adult-onset minimal change disease. Nephrol Dial Transplant 2008;23:169-75.  Back to cited text no. 22
    
23.
Haack D, Schärer K, Asam-Tauscher A, Vecsei P. Glucocorticoid receptors in idiopathic neph-rotic syndrome. Pediatr Nephrol 1999;13:653-6.  Back to cited text no. 23
    
24.
Ali SH, Ali AM, Najim AH. The predictive factors for relapses in children with steroid-sensitive nephrotic syndrome. Saudi J Kidney Dis Transpl 2016;27:67-72.  Back to cited text no. 24
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25.
Vivarelli M, Moscaritolo E, Tsalkidis A, Massella L, Emma F. Time for initial response to steroids is a major prognostic factor in idiopathic nephrotic syndrome. J Pediatr 2010; 156:965-71.  Back to cited text no. 25
    
26.
Zahran AM, Aly SS, Elsayh KI, Badawy A, Gamal Y. Glucocorticoid receptors expression and histopathological types in children with nephrotic syndrome. Ren Fail 2014;36:1067-72.  Back to cited text no. 26
    

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Correspondence Address:
Mohamed A El-Gamasy
Department of Pediatrics, Faculty of Medicine, Tanta University, Tanta, Gharbia
Egypt
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DOI: 10.4103/1319-2442.239659

PMID: 30152427

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