The Clinical Profile and Long-Term Outcome of Children with Membranous Nephropathy, and the Evaluation of Anti-Phospholipase A2 Receptor Antibody Immunohistochemistry in Kidney Biopsy

Patient selection

This 15-year retrospective study was conducted in the pediatric nephrology division at a tertiary care hospital in South India. Children below 16 years who were diagnosed as MN by kidney biopsy from Jan 2005 to Oct 2020 were included. Anti PLA₂R IHC was performed in the archived kidney biopsy specimens. The objective was to evaluate the clinical profile and long-term outcome of both iMN and SMN and assess the sensitivity of PLA₂R IHC in children with iMN. Institutional research board and ethical committee clearance was obtained. Waiver of consent was sought since retrospective analyses of archived renal biopsy specimens were performed and data were collected from electronic health records.

Clinical details with definitions

Demographic and biochemical data were collected from electronic records and collected at onset, quarterly till 12 months, and thereafter at 2, 5, and 10 years. Secondary causes of MN were ruled out by performing hepatitis B virus surface antigen (HBsAg), human immunodeficiency virus (HIV), hepatitis C, anti-nuclear antibody, chest X-ray, ultrasound abdomen, and stool occult blood in necessary cases.

NS was defined as per the kidney disease: improving global outcomes (KDIGO) guidelines 2012.[22] The Toronto risk score is not feasible in children since all childhood-onset NS cases receive corticosteroids before kidney biopsy unlike adult NS.[23] Hypertension (HTN) was defined as blood pressure above 95^th percentile for age, gender, and height as per the Fourth Report.[24] Estimated glomerular filtration rate (eGFR) was obtained using the modified Schwartz’s formula.[25] Microscopic hematuria was defined as more than five red blood cells (RBCs) per high-power field of centrifuged urine sample.

Complete remission (CR) was defined as urine protein nil or trace and urine protein creatinine ratio (uPCR) <0.3 mg/mg with a normal serum albumin and creatinine; partial remission (PR) was defined as uPCR <3.5 mg/mg and a 50% or greater reduction from baseline with a stable eGFR and serum albumin >3.5 g/dL. Nonresponse was lack of CR or PR after 6 months of therapy. Relapse was defined as recurrence of proteinuria after being in remission previously.[26,27] The choice of the immunosuppressive agents was as per the discretion of the treating physician and was based on patient characteristics, previous treatment, side effect profile, affordability of drugs, and parental preference.

Histopathology

Kidney biopsy samples were fixed in 10% neutral buffered formalin and embedded using standard methods. Tissue for light microscopy consisted of serial 3-μm-thick sections, stained with hematoxylin and eosin, periodic Schiff reagent, Masson trichrome, and Jones methanamine silver stains. Immunofluorescence studies consisted of 4 μm cryostat sections stained with fluorescein-tagged polyclonal rabbit anti-human antisera specific to IgG, IgA, IgM, complement factors C3, C1q, and to kappa and lambda light chains (all from Dako, Carpinteria, CA, USA).

IHC staining for anti-PLA₂R antibody (Sigma Aldrich) was performed on “Ventana Benchmark XT autostainer” multimer method with diaminobenzidine. Dilution of the primary antibody used was 1:1000. Positive IHC staining of anti-PLA₂R antibody was defined by the complete staining of the glomerular capillaries. All our cases showed either strong PLA₂R positive or absent staining of glomerular capillary walls, as shown in Figure 1.

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Figure 1

(a) Granular staining for PLA2R1 along glomerular basement membranes in a patient with idiopathic membranous glomerulopathy (immunohistochemistry; original magnification 400). (b) Glomerulus from patient with idiopathic membranous glomerulopathy is completely negative for PLA2R1 (immunohistochemistry; original magnification 400).

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Statistical analysis

Data were presented as the mean ± standard deviation or median (interquartile range [IQR]) depending on the normality of distribution. Student’s t-test and analysis of variance (ANOVA) were used for parametric analysis. Comparison of means was done using independent samples t-test. Correlation between two parameters was assessed using Pearson coefficient. Statistical significance was considered when P < 0.05. Statistical analysis was done using IBM Statistical Package for the Social Sciences (SPSS) Statistics Version 21 (IBM Corp., Armonk, NY, USA)

Patient characteristics

MN constituted 3.2% of pediatric kidney biopsies over 15 years. They were further divided into iMN in 42% (n = 18) and SMN in 58% (n = 25). Figure 2 depicts the flow diagram of the study indicating enrolment and follow-up. In the study group, the incidence of MN increased as age progressed, with 7% of MN in 1–5 years, 23% in 6–10 years, and 70% in 11–15 years. In children below 10 years of age, SMN was significantly more than iMN (69%, P = 0.04). At the initial presentation, nephrotic proteinuria was present in 90.7% (n = 39) with uPCR of 6.9 (3.4, 11.2), microhematuria in 95% (n = 41), and hypertesion in 25% (n = 11) of cases. The mean eGFR at the onset was 156 ± 81 ml/min/1.73m². NS was present in 54% (n = 23/43) patients. The demographic features are presented in Table 1. The presentation in childhood iMN was late-onset NS and steroid-resistant NS in eight patients each (44.4%) and nephritic-onset NS in two (11%) patients. Most common causes of SMN were lupus nephritis (16, 64%), hepatitis B (8%), infection-related glomerulonephritis (8%), non-lupus full house nephropathy (8%), graft versus host disease post bone marrow transplant (4%), celiac disease with sarcoidosis and chronic liver disease (4%), and tuberculosis (4%). All the biopsies were reviewed and none of the biopsies had significant chronic changes. Only few patients showed chronic changes at the time of biopsy, that is, minimal (0–5%) interstitial fibrosis/tubular atrophy with <5% glomerulosclerosis and no evidence of arteriosclerosis/arteriolar hyalinosis. Children with PLA₂R-negative iMN had higher degree of proteinuria (18.4 vs. 15.6, P = 0.84) and higher incidence of HTN (43% vs. 14, P = 0.00).

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Figure 2

Flow diagram of the study patients showing enrolment and follow up of 43 patients from 2005 to 2020

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Treatment received

40% (n = 7/18) of iMN and 28% (n = 7/25) of SMN children received only corticosteroids. Mycophenolate mofetil was added in one-third (n = 6/18, 33.3%) of iMN and more than half (n = 14/25, 56%) of SMN. Third most common immunosuppressive agent used was tacrolimus (12%). The details of treatment received have been summarized in Table 2. There was no significant difference in the clinical outcomes between the different immunosuppressive agents.

Response to therapy and outcome

Mean time to CR in iMN and SMN was 14 (4.5, 34.5) and 8.5 (3.8, 14.3) months, respectively (P = 0.13). The proportion of patients with CR/PR at the last follow-up was 86% (n = 12/14) in iMN and 90.9% (n = 20/22) in SMN (P = 0.59). Figure 3a shows the Kaplan–Meier graph depicting the response rate among the patients with iMN and SMN, showing that SMN had better remission rates and iMN took longer time for remission, though the difference was not statistically significant. Number of patients who had persistent proteinuria was significantly more in iMN (55.5% vs. 23%) in the first year and at the final visit (93% vs. 4.6%) (P = 0.01). iMN had more relapses (0.44 ± 0.98) than SMN (0.24 ± 0.58) (P = 0.38). CKD 5 developed in 2/14 (14.3%) iMN patients and both were PLA₂R positive. The long-term follow-up and outcome of children with MN is presented in Table 3. Response to therapy assessed as CR or PR at various intervals is depicted in Figure 4. Children with PLA₂R-negative iMN had higher degree of proteinuria (18.4 vs. 15.6, P = 0.84) and higher incidence of HTN (43% vs. 14%, P = 0.00). The time taken to achieve PR was more in PLA₂R-negative children (5.7 ± 6.3 months) than PLA₂R-positive children (2 ± 3.3 months), and none of the PLA₂R-negative iMN had attained CR. PLA₂R-negative children had higher persistent proteinuria at the end of therapy. Figure 3b depicts the response rate among the patients with PLA₂R-positive and -negative iMN. Though PLA₂R-negative iMN children were noted to have more severe clinical presentation at the onset and took longer time to respond to treatment, there were no statistically significant differences between the two groups on long-term follow-up in terms of remission. The details of PLA₂R-positive and -negative subgroups are given in Supplementary Tables 1–3.

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Figure 3

(a) Mean time to complete remission in iMN and SMN were 14 (4.5, 34.5) and 8.5 (3.8, 14.3) months respectively (p=0.13). The proportion of patients with CR/PR at last follow up was 86% (n=12/14) in iMN and 90.9% (n=20/22) in SMN (p=0.59). (b) There were no statistically significant differences between the two groups who were PLA2r positive and negative on long term follow up in terms of partial or complete remission

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Figure 4

Response rates were measured by uPCR at various intervals of follow up and values expressed in percentages. CR: complete remission, PR: partial remission, NR: non-remission, eGFR estimated GFR

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As shown in Table 4, we performed univariate logistic regression with outcome parameters for assessing the risk factors for nonresponse at 6 months. The regression analysis did not show any significant association with outcome.

Limitations

Though we have described a significant number of children (n = 43) with MN followed from a single center in India, it is truly difficult to conclude and generalize these findings with the present numbers. PLA₂R staining could not be done in all the kidney biopsy specimens of iMN as paraffin blocks could not be retrieved from the archives. Serum anti-PLA₂R evaluation was possible only in some patients, and hence, their data was not summarized. In view of the small number of patients in each subgroup, multivariate regression analysis could not be performed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.