The expression of cytoskeletal proteins in kidney specimens of children with primary focal segmental glomerulosclerosis

Patients

This historical cohort study was carried out in Isfahan, a large central province of Iran. In the time period between November 2007 and April 2010, we consecutively recruited 31 children with primary FSGS, who were referred to pediatric nephrology wards of the Isfahan University of Medical Sciences, the main health support organization of the region. The inclusion criteria were (i) age at onset of 1-18 years (ii) definit diagnosis of primary FSGS with renal biopsy, (iii) adequate kidney tissue in paraffin-embedded specimens to prepare new slides for immunohistologic staining, and (iv) follow-up period of at least two years for each case. The histopathologic diagnosis of FSGS was based on the following criteria: (i) lesion affecting some of the glomeruli in the renal biopsy whereas others remain unaffected and (ii) the affected glomeruli having a portion that has undergone capillary collapse with obliteration of capillary lumina with or without adhesions. It is worth noting that cases with clinical or pathologic evidence suggestive of secondary FSGS (e.g., reflux nephropathy, renal hypodysplasia, severe obesity, cyanotic heart disease, and unilateral renal agenesis) and also those with congenital forms of the FSGS were not included in this study.

Demographic, clinical, and laboratory data of eligible cases were collected retrospectively by review of medical records. These data comprised height, weight, age at onset, 24-h urinary protein excretion, serum albumin, serum creatinine, creatinine clearance (based on Schwartz formula), and systolic and diastolic blood pressure. It must be noted that these data were updated regularly (every 3-6 months), during follow-up visit for each patient.

Definitions

Chronic kidney disease (CKD) was defined as glomerular filtration rate (GFR) lower than 90 ml/min/1.73m². Hypertension was defined as blood pressure higher than 95^th percentile for age and height according to data from the Task Force Report on High Blood pressure in Children and Adolesents.[22] Response to treatment was classified as complete remission, partial remission, and non-remission: Complete remission was defined as negative or trace proteinuria adjusted by Body Surface Area <350 mg/m²/day. Partial remission was defined as a reduction in proteinuria but still remaining in the supranormal range. Non-responder (steroid resistant nephrotic syndrome [SRNS]) was considered as inability to induce remission and/or partial remission within 4 weeks of daily steroid therapy followed by three pulses of methyprednisolon. Steroid sensitive nephrotic syndrome (SSNS) was defined as remission after 4-6 weeks of full dose of steroid therapy.

Treatment protocols

All patients underwent similar initial treatment strategy. They were initially treated with oral 60 mg/m²/day prednisolone for 4-6 weeks, with or without three consecutive doses of methyl prednisolone pulses (10-30 mg/kg/dose). This was then tapered in an alternate-day regimen over six to nine months.

Patients who responded partially to prednisolone or demonstrated steroid side effects received cyclophosphamide (2-3 mg/kg/day for 2-3 months). For those patients who did not respond to aforementioned medications, cyclosporine A (3-5 mg/kg/day) was commenced after performing kidney biopsy. In cases that did not respond to cyclosporine A in a 6-month course, mycophenolate mofetil (500-1,000 mg/m²/day) was replaced.

Angiotensin converting enzyme inhibitor (ACEI) was added as an adjuvant therapy to control hypertension or proteinuria.

Tissue specimens and immunohistochemistry

A total of 31 formalin-fixed, paraffin-embedded kidney biopsy sections (3 μm) were used for immunohistochemical staining. The following monoclonal antibodies (mAb) were used: (i) Monoclonal mouse anti-SMA1 antibody (Dako, Clone 1A4, Code M0851) at a running dilution of 1:50. Studies have generally accepted its specificity for smooth muscle actin.[23] (ii) Monoclonal mouse anti-human vimentin antibody (Dako, Clone V9, code M 0725) at a running dilution of 1:200. This antibody is commonly used to stain glomerular epithelial cells.[24] (iii) Monoclonal mouse anti-human desmin antibody (Dako Clone D 33, code M 0760) at a running dilution of 1:50. It has shown specificity for staining glomerular epithelial cell in rats.[25]

Double immunohistochemistry, using a microwave-based two color staining was applied.[26] Tissue sections were baked, dewaxed, hydrated to distilled water, and then pre-incubated in 10% FCS and 10% normal goat serum to block non-specific binding. After applying primary antibodies (anti α-SMA, desmin and vimentin m-Ab), the samples were incubated for 30 minutes at room temperature. Then the slides were washed two times in Tris Buffer Saline (TBS) and quenched with 0.3% H₂O₂ in methanol for 10 min to block endogenous peroxidase. The sections were incubated for 30 min after applying secondary antibody (goat anti-mouse IgG, DAKO). After rinsing 3 times with TBS, mouse PAP in a dilution of 1/50 was used. These steps were followed using diaminobenzidine (DAB) for 5 min to produce a light brown color. The slides were rinsed in tap water for 5 min. Finally, the slides were counter-stained with hematoxylin (blue) and then dehydrated and mounted with entelan glue.

Quantitation of tissue staining

All of the specimens were reviewed by a pathologist who was blinded to the clinical features and outcome of patients. The immunohistochemical staining was estimated stereologically by a graticule provided 121 points each field that was superimposed upon the specimen.

For each specimen, the following features were recorded:

1. Scoring for tubular staining with mAB in each tubular cross-section:

   • 0 epithelial tubular cell stained =0

   • 1-2 epithelial tubular cells stained = +1

   • >2 epithelial tubular cells and less than 50% of cells stained = +3

   • More than 50% of epithelial tubular cells stained = +4

2. At least 100 tubules and at most 500 tubules were scored and the average was reported as the final score:

   Scoring for interstitial staining with monoclonal antibodies after overlaying an ocular grid:

   • Each field (grid) =121 points

   • A = number of positive points that matched completely with positive cells

   • B = number of non-scored points

   • C = number of assessed points (121-B)

   • D = percentage of positive points = A/C

   • Total score = D1+D2+…+Dn/total number of fields

Statistical analysis

Statistical analyses were performed using SPSS for Windows (version 16). The categorized data were reported as frequencies and percentages. Continuous data were reported as mean and standard deviation (SD). The renal survival was evaluated using the Kaplan-Meier method. To determine the predictive value of each variable for the outcome, the Cox regression was used. Log rank test was used to compare renal survival between steroid responders and non-responders and also among patients reaching complete remission and partial remission. Pearson's correlation test was used to analyze correlations. In addition, we used receiver operator characteristic curve analysis to detect the best cut-off values for cytoskeletal proteins (vimentin and α-SMA) to predict the outcome. A P value lower than 0.05 was considered as a significant threshold.

Ethics

Written informed consent from all parents and oral assent from children above six years of age were obtained for this study. This survey was performed in accordance with the Helsinki Declaration.

Clinical features

In this study, 16 (51.6%) and 6 (19.3%) cases reached complete and partial remission respectively. Cumulative incidence of remission was 22 (70.9%). However, five patients (16.1%) did not reach remission during the observation period. In addition, the majority of our patients were categorized as non-responder to steroid therapy (67.7%).

Twenty six out of 31 patients (83.8%) received angiotensin receptor blockers (ARBs) or ACEI for treatment of hypertension and/or to decrease the amount of proteinuria.

Further laboratory and clinical features of the patients, both in the time of presentation and in our final evaluation, are summarized in Table 1. The results of analysis (Inter model) revealed that response to steroid and the last diastolic blood pressure were predictive variables for the last GFR (P < 0.05).

Table 1 Clinical characteristics of the patients participated at the study

Renal survival

Among the patients, 51% progressed to CKD. The 1-year and 5-year renal survival rates in our study were 86.7% and 41.7%, respectively [Figure 1]. These rates were not significantly different between two sexes (P > 0.005). The median renal survival was 60 ± 29.5 months.

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Figure 1

Median Renal Survival in children with focal segmental glomerulosclerosi

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The median of renal survival in steroid responders (33.3% of patients) and non-responders (66.6% of patients) were 161 ± 10.2 and 25 ± 3.05 months, respectively [Figure 2].

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Figure 2

Renal survival according to response to steroid

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Histopathology

The mean number of assessed glomeruli (in each specimen) was 16.5 ± 9.6. Interstitial fibrosis and tubular atrophy were reported in 42% and 54% of patients respectively. Almost all patients who had interstitial fibrosis were also reported to have tubular atrophy. However, vessel changes only were reported in three patients (9.6%). In the non-responder patients, Multiple Regression Analysis showed interstitial fibrosis as the only histopathologic predictor of non-response to steroid and diastolic and systolic hypertension (P < 0.05).

Immunohistochemistry findings

Light microscopic and immunohistochemical staining for vimentin and α-SMA are shown in Figure 3. In stained specimens, mean vimentin expression score for interstitium and tubules were 0.19 ± 0.17 and 2.24 ± 0.78 respectively.

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Figure 3

(a) Focal segmental glomerulosclerosis (FSGS), light microscopic examination showing collapse of capillaries together with mesangial matrix expansion (masson trichrome staining, original magnification, ×400). (b) Staining for vimentin, using an anti-vimentin antibody, showing an increase in interstitium of a case with FSGS (original magnification, ×400). (c) Immunostaining for vimentin showing positivity in tubular epithelial cells of a patient with FSGS (original magnification, ×400). (d) Positive α SMA immunostaining in mesangial and epithelial cells of a FSGS glomeruli (original magnification, ×400)

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The mean score of vimentin and α-SMA expression in interstitium were not significantly different between patients who had GFR lower or greater than 90 ml/min. Similar results were obtained considering GFR lower or greater than 70 ml/min and 50 ml/min (the results are not shown).

The mean point counting scores for α-SMA were 6.17 ± 5.97 and 0.27 ± 0.13 for glomeruli and interstitial respectively. Desmin was not detected in any of the tubular, interstitial, or glomerular staining. Tubular vimentin expression was correlated with systolic blood pressure at presentation (P = 0.01, r = 0.46). Interstitial vimentin expression was correlated with systolic blood pressure at presentation and also systolic and diastolic blood pressure at last visit (r¹ = 0.37, r² = 0.4, r³ = 0.5 respectively). Interstitial α-SMA expression was only correlated with the last evaluated diastolic blood pressure (P = 0.015, r = 0.46). α-SMA interstitial staining was correlated with vimentin interstitial staining (P = 0.010, r = 0.478). The last evaluated GFR was only correlated with tubular expression of vimentin, P < 0.05. These findings were approved by regression analysis, as well.

However, multiple regression analysis did not show any significant prognostic value for the aforementioned pathologic factors.