Differential alteration in peripheral T-regulatory and T-effector cells with change in P-glycoprotein expression in Childhood Nephrotic Syndrome: A longitudinal study

Highlights

• Frequency of Tregs are increased during remission and decreased at relapse.

• Frequency of effector T cells are decreased during remission and increased at relapse.

• Cytokines IL-10 and TGF-β from PBMCs are increased during remission and reversed at relapse.

• P-gp expression remains greater in relapsing than non-relapsing patients during remission.

• Longer steroid therapy is required to achieve remission in patients with higher P-gp expression.

Abstract

Introduction

Childhood Idiopathic Nephrotic Syndrome (INS) responds to glucocorticoid therapy, however, 60–80% of patients relapse and some of them become steroid non responsive. INS may occur because of T cell dysfunction, abnormal cytokines and podocytopathies which reverse on steroid treatment. The reason of relapses could be imbalances in T cells phenotypes and respective cytokines. Herein, we hypothesize that relapses in INS may occur due to imbalance in T-regulatory and T-effector cell with their respective cytokines and overexpression of P-gp on lymphocytes.

Methods

The frequency of peripheral blood CD4⁺CD25⁺FoxP3⁺ Treg, CD4⁺IFN-γ⁺ Th1 and CD4⁺IL-4⁺ Th2 lymphocytes and their respective cytokines and P-gp expression on peripheral blood lymphocytes (PBLs) were analyzed in INS patients at baseline (n = 26), during remission (n = 24) and at relapse (n = 15).

Results

Compared to baseline, the frequency of Tregs was significantly increased at remission and decreased during relapse. In contrast, the frequency of Th1 and Th2 lymphocytes was significantly decreased during remission and increased at the time of relapse. Similarly, expression of P-gp was significantly high at baseline and at the time of relapse as compared to remission. Levels of cytokines IL-10 and TGF-β in the supernatant of stimulated PBMCs was increased during remission and decreased during relapse. In contrast, levels of IFN-γ and IL-4 were decreased during remission and increased at the time of relapse.

Conclusions

Steroid therapy in INS induces decreased P-gp expression on PBLs along with increased frequency and cytokine response of T-regulatory cells, and reduced frequency and respective cytokine response of Th1 and Th2 cells during remission. However, reversal in the frequency and respective cytokines of T-regs, Th1 and Th2, and P-gp expression on PBLs occurs during relapses on follow-up.

Introduction

Minimal change disease (MCD) is one of the most common glomerular diseases causing Idiopathic Nephrotic Syndrome (INS) in children. MCD may be associated with T-cell dysfunction [1], increase in cytokines level [2], [3], [4] and podocyte injury which may be reversed after steroid therapy [5], [6]. The exact cause of this podocytopathy in MCD remains unknown. Podocytopathy in MCD may be idiopathic, genetic; associated with NPHS2 mutation or reactive associated with malignancy or other immunologic stimuli. Non genetic idiopathic and reactive forms of MCD are usually steroid responsive and cell mediated immunity has been invoked as etiologic factor for this form of MCD [1], [5]. However the predominant role of different T cell phenotypes; regulatory T cells (Tregs), and effector T cells (Teff) T helper cells (Th1) and T helper cells (Th2) remain poorly understood [7]. The predominance of Th2 phenotype was reported in one study while another study did not report any skewing of Th1/Th2 and suggested a role of Tregs [8]. Araya et al have shown that suppressor function of Tregs cell was deficient in INS that could lead to persistence of pathogenic cytokines released by Teff cells resulting into proteinuria [9]. A case of INS going in to remission following influenza B infection without the need of steroids has been reported. Authors have demonstrated increased Tregs population during remission in the case [10]. Recently, we have observed greater ratio of Tregs/Effector (Teff) cells in remission in INS patients and its reversal during treatment resistant state [11].

Approximately 60–80% of steroid responsive INS patients experience relapses of proteinuria on follow up despite initial clinical response to steroids and some of them become steroid non responsive [12]. The treatment of relapsing Nephrotic Syndrome with multiple courses of steroids, cytotoxic agents and calcineurin inhibitors remains a challenging clinical scenario [13]. The reason of poor steroid response could be change in histological pattern from MCD, a condition with podocyte injury without podocytopenia, to FSGS podocyte injury with podocytopenia [14], or changes in pharmacological intervention with overexpression of permeable glycoprotein (P-gp) on lymphocytes which has emerged as one of the major molecule causing poor response to many drugs, peptides, alkaloids, steroids, immunosuppressive drugs, and calcium channel blockers [15]. P-gp is a 170-kD product of the multidrug resistance 1 (MDR-1) gene. Our previous study indicated that homozygous mutant of MDR-1 gene influences steroid responsiveness in NS patients [16]. The MDR-1 gene belongs to the ATP-binding cassette (ABC) energy-dependent transporters [17]. In humans, P-gp is involved in xenobiotics efflux, protecting host tissue from toxic side effects. The overexpression of P-gp causes efflux of steroid from inside of cells to the outside. This limits the concentration of steroids within the cells and their site of action that results in poor response and steroid resistance [18]. P-gp is expressed on the surface of peripheral blood lymphocytes (PBLs), which are the putative targets of pharmacotherapy in INS [19] and the expression of P-gp is regulated by certain cytokines at transcription level which plays role in pathogenesis of INS [20].

Corticosteroids and calcineurin inhibitors are the drugs used for the treatment of NS. It is possible that podocyte itself could be the target of steroid or calcineurin inhibitors in INS as receptors of these drugs has been seen on podocytes. The effect of corticosteroids on lymphocytes and cell mediated immunity in INS is well established and the effect of steroid therapy on PBLs and phenotypic changes in patients during the course of the therapy is easier to monitor than any changes on podocytes. Corticosteroids are the substrate of P-gp and calcineurin inhibitors are substrate as well as inhibitor of P-gp expressed on lymphocytes [19]. The many poor steroid responsive INS patients respond well to calcineurin inhibitors. There is paucity of data on this novel biomarker during the course of the disease. It may help in better monitoring of the disease status. Therefore, we aimed this study to evaluate the alterations in frequency of different peripheral blood T cell phenotypes; Tregs, Th1, Th2 cells, and P-gp expression on PBLs in INS patients receiving steroids at baseline, on achieving remission and at time of relapse.