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Rituximab in Minimal Change Disease

Treatment with rituximab, a monoclonal antibody against the B-lymphocyte surface protein CD20, leads to the depletion of B cells. Recently, rituximab was reported to effectively prevent relapses of glucocorticoid-dependent or frequently relapsing minimal change disease (MCD). MCD is thought to be T-cell mediated; how rituximab controls MCD is not understood. In this review, we summarize key clinical studies demonstrating the efficacy of rituximab in idiopathic nephrotic syndrome, mainly MCD. We then discuss immunological features of this disease and potential mechanisms of action of rituximab in its treatment based on what is known about the therapeutic action of rituximab in other immune-mediated disorders. We believe that studies aimed at understanding the mechanisms of action of rituximab in MCD will provide a novel approach to resolve the elusive immune pathophysiology of MCD.


Introduction
Primary nephrotic syndrome, albeit rare, is the most common chronic glomerular disorder in childhood. The vast majority of children presenting with nephrotic syndrome will have minimal change disease (MCD), a clinical and pathological entity characterized by nephrotic-range proteinuria, hypoalbuminemia, hypercholesterolemia, and absence of glomerular immune deposits or cellular infiltrates in the biopsy. 1 The sole histological abnormality is the disappearance of podocyte foot processes (effacement) that is detected by electron microscopy. 1 MCD can also develop de novo in adulthood. The pathogenesis of MCD is not known, but it is believed to be T-cell mediated. [1][2][3] Pediatric MCD is generally responsive to glucocorticoids. As a kidney biopsy is reserved for patients with a complicated or treatment-recalcitrant course, 4 the term MCD is often used interchangeably with glucocorticoid-sensitive nephrotic syndrome (often referred to as steroid-sensitive nephrotic syndrome or SSNS) or idiopathic nephrotic syndrome (INS). However, up to 50% of children with INS experience frequent relapses or glucocorticoid dependence, known as frequently relapsing (FRNS) or steroid-dependent nephrotic syndrome (SDNS). 5 Moreover, about 10% to 20% of the patients show primary or late glucocorticoid resistance (steroid-resistant nephrotic syndrome [SRNS]). 5,6 Long-term glucocorticoid use in FRNS/SDNS patients leads to co-morbidities such as cushingoid habitus, growth retardation, striae and acne, reduced bone mineral density, cataracts, pseudotumor cerebri, hypertension, impaired glucose tolerance, hypercholesterolemia, and increased infection risks. 7 Hence, alternative (glucocorticoid-sparing) medications have been introduced. Commonly used "second-line" drugs are oral alkylating agents (mainly cyclophosphamide), calcineurin inhibitors (CNIs, cyclosporine and tacrolimus), levamisole, and mycophenolate mofetil. 4,8 However, these medications are not uniformly effective in suppressing relapses of proteinuria and are associated with their own spectrum of adverse effects.
During recent years, rituximab (RTX) has been used successfully as a novel treatment modality in patients with INS/ MCD. RTX is a chimeric monoclonal antibody containing murine variable regions and a human IgG1 constant domain against CD20. CD20 is a membrane protein expressed on the surface of B lymphocytes, with the exception of late pro-B and plasma cells, and appears to play a role in intracellular Ca ++ influx and activation of B cells, and mediates cellular proliferation and differentiation of B cells. RTX causes the elimination of CD20 + B cells by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct induction of apoptosis. 9 B-cell suppression lasts a few months, but can vary substantially. How RTX works in MCD is not understood and indications for its administration compared with other second-line agents remain to be defined.
The goal of the current review is to explore the mechanisms, by which RTX works in MCD. We will examine the clinical literature on the use of RTX in MCD/INS in children and adults and review available data on the immune pathogenesis of MCD. Finally, we will synthesize hypotheses on the potential mechanisms of action of RTX in MCD, based on the pathophysiology of MCD and what is known about the therapeutic action of RTX in other immune-mediated diseases. We believe that studies aimed at understanding the mechanisms of action of RTX in MCD will provide a novel approach to study the elusive immune pathophysiology of MCD.

RTX in Children With SDNS/FRNS
In addition to small case series or specific case scenarios, [10][11][12][13][14] we identified 4 randomized, controlled trials (RCTs) from 2 centers examining the efficacy and short-and long-term outcomes of RTX treatment in children with glucocorticoid responsive INS (summary in Table 1). The Rituximab for Childhood-Onset Refractory Nephrotic Syndrome Study Group performed a multicenter, double-blind, randomized, placebo-controlled trial at 9 centers in Japan on 48 children with FRNS and SDNS. The reported median relapse-free period was significantly longer in the RTX group than in the placebo group. The authors concluded that RTX is an effective and safe treatment for childhood-onset FRNS and SDNS. 15 Ravani et al conducted an open-label RCT comprising 54 children with glucocorticoid-dependent and CNIdependent INS in Italy. The authors noted that RTX along with reduced doses of prednisone and CNI is non-inferior to standard full-dose prednisone plus CNI treatment in maintaining short-term remission. 16,17 In another RCT by Ravani et al, 18 3-month proteinuria was non-inferior in the RTX plus tapered prednisone group compared with the prednisoneonly group and median time to relapse was longer in the RTX group. Kidney biopsies were not required or reported in any of the reported trials.
Ruggenenti et al performed a multicenter, off-on trial of RTX in 10 children and 20 adults with different pathologies of INS. All pediatric INS cases were glucocorticoid dependent. Comparing 1-year follow-up after RTX with the year before RTX treatment, per-patient median number of relapses, prednisone maintenance dose, and the median cumulative dose of glucocorticoids to maintain remission were significantly decreased. 19 Overall, there is strong evidence to support the efficacy of RTX in glucocorticoid responsive MCD/INS in children. and CNI for a month, when they started tapering both medications if proteinuria had decreased to < 1 g/day/ m 2 RTX plus standard treatment failed to induce remission in those who had been previously unresponsive to a combination of CNI and prednisone despite target drug serum levels achieved and CD20 counts despite target drug serum levels and CD20 counts achieved. Three children in each group, all with late resistance to prednisone and alternative agents, entered remission. No subject with primary treatment resistance entered remission in either arm

RTX in Adults With SDNS/FRNS
RTX appears to be beneficial also in adults with FRNS/ SDNS MCD. Takei et al performed a prospective trial of 25 adults with SDNS and observed that RTX treatment was associated with a reduction in the number of relapses and the total dose of prednisolone needed. The authors concluded that the efficacy of RTX in the treatment of adult SDNS is similar to that in childhood MCD. 20

RTX in Patients With SRNS
Early case reports suggested that RTX could be a promising agent also in SRNS. However, in contrast to the demonstrated efficacy of RTX in the treatment of SSNS, results in patients with SRNS were inconsistent. Gulati et al studied a largely pediatric cohort involving 33 patients with SRNS from 2 academic tertiary centers in India and the United States. 23 Six months after the infusion of RTX, with continued (reduced) immunosuppressive therapy, 27% of the SRNS patients were in complete remission, 21% had partial remission, and 51% failed to respond. A questionnaire-based retrospective study of 70 patients from 25 international centers reported a response rate to RTX of 82%, 44%, and 60% for SDNS/FRNS, SRNS, and recurrent FSGS post-transplant, respectively. 24 In another survey of 70 patients in Japan, 77% (SDNS/FRNS) and 29% (SRNS) patients successfully discontinued prednisone, the majority of them for the first time since disease onset. However, 51% of these patients relapsed. 25 Other, smaller case series reported variable response rates of SRNS to RTX. [26][27][28] In a series of 4 children with SRNS, none of the patients achieved sustained remission after a single dose of RTX, despite effective B-cell depletion. 29 Throughout these studies, the response rate to RTX was consistently lower in SRNS than in SDNS/FRNS, and in most instances, SRNS patients received additional, concomitant immunosuppressants. An open-label multicenter RCT of 31 children with INS showed that 2 doses of RTX given in addition to standard treatment failed to induce remission in those who had been previously unresponsive to a combination of CNI and prednisone. 30 No subject with primary treatment resistance entered remission in either arm. The authors concluded that RTX should not be considered in children unresponsive to glucocorticoids and CNI, especially in those with primary or early unresponsiveness. Similarly, Fernandez-Fresnedo et al reported that none of 8 adults with SRNS and a histological diagnosis of FSGS achieved complete or partial remission of nephrotic-range proteinuria following 4 doses of RTX with concomitant immunosuppressive therapy. 31 Thus, RTX, alone or in combination with other immunosuppressants, cannot be recommended to induce or maintain remission in patients with SRNS. 32,33 Variable response rates to RTX reported among patients with SDNS/FRNS and SRNS are likely due to heterogeneity in the pathogenesis of "idiopathic" nephrotic syndromes. Tissue diagnosis is missing in some series, especially in glucocorticoid and CNI-responsive patients. Some authors noted a poor correlation between the biopsy diagnosis and responsiveness to RTX. 24 Comprehensive genetic testing is rarely available, potentially leading to the inclusion of patients with undiagnosed podocyte gene mutations associated with a lack of response to immunosuppressive therapy. 34 Additional genetic factors may modify the responsiveness to RTX. Examples are polymorphisms in genes encoding FcγR, [35][36][37][38] interleukin (IL)-6, 39,40 transforming growth factor (TGF)-β, 41 B-lymphocyte stimulator (BLyS), also known as B-cell activating factor (BAFF), 42,43 and type I interferon, as shown in patients with rheumatoid arthritis (RA). 44,45 Genetic variants may cause incomplete B-cell depletion or unresponsiveness to treatment despite complete B-cell depletion. Loss of RTX in the urine when administered during active nephrotic syndrome is a consideration. 46,47 Early (peak) RTX serum levels 24 hours post-infusion were not influenced by the degree of proteinuria, 30 practically excluding differences in B-cell elimination efficacy; however, proteinuria will likely impact on the duration (maintenance) of B-cell suppression.

Possible Mechanisms of Action of RTX in MCD
As noted above, there is strong clinical evidence that RTX effectively suppresses relapses of proteinuria in patients with glucocorticoid-sensitive MCD. Clinical observations and earlier laboratory studies suggest that the biological changes in MCD are caused by systemic immunological dysregulation and that T cells are the main effector in the process leading to podocyte foot process effacement and resultant heavy proteinuria. [1][2][3] The therapeutic efficacy of B-cell depleting agent not only challenges this dogma, but also affords an opportunity to approach the pathogenesis of MCD from a new angle. In healthy individuals, B cells not only secrete antibodies, but they also shape protective T-cell responses by providing antigen and co-stimulatory signals, and also produce cytokines that modulate T-cell differentiation. B cells are effector cells in their own right as well. 48 In MCD, post-RTX relapses often occur with B-cell recovery, likely an indication of a pathogenic B-cell population resurgence. 49 Because autoantibodies are not a feature of MCD, B-cell functions other than antibody production should be explored in the quest of understanding how RTX modulates MCD. Studies focusing on the mechanisms of action of RTX in MCD are limited to the description of B cells during the recovery phase following B-cell depletion, 49 or postulated direct effects of RTX on podocytes. 50 We will discuss possible mechanisms of RTX's actions in MCD based on current knowledge of its effects in other T-cell-mediated diseases and of the immune pathophysiology of MCD. In Table 2, we reviewed and summarized studies addressing the mechanisms of action of RTX in recognized autoimmune diseases, such as RA, systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpura (ITP), multiple sclerosis (MS), and type 1 diabetes mellitus (DM1) focusing on non-antibody-mediated aspects of these diseases.

RTX-Associated Change in the Absolute or Relative Quantity of T-Cell Subsets and B Cells
T cells are divided into subsets with distinct immunological profiles such as CD4 + T helper (Th) cells, which generally  [51][52][53] Another study failed to observe T-cell depletion 54 55 3. Decreased RANKL expression in synovium and serum 56 suggesting inhibition of Th cell activity 4. Decreased Mϕ counts and activity 53,55,57,58 5. Decreased cytotoxic lymphocytes and mature NK cells and a simultaneous upregulation of activated NK cells 59,60 6. Decreased IL-15 and IL-17. Clinical improvement may be associated with IL-15/memory T-cell-related mechanisms. 61 (IL-15 is known to expand and activate NK cells.) 7. Decreased IL-2, IL-6, IL-7, and IL-10 serum levels 62 8. Decreased production of IFN-γ and IL-1β by T cells 55 9. Downregulation of genes involved in immunoglobulins, chemotaxis, leukocyte activation, and immune responses with upregulation of genes involved in TGF-β pathway in synovial tissue 63  1. Although anti-T-cell therapies (eg, anti-CD3 antibody) are effective in treatment of DM1, 85 RTX was also shown to be helpful in new-onset DM1 86 2. Reduction in autoantibody production or autoreactive B-cell counts 87 3. Increased Treg cells in short term 86 4. Modification of the B7-2/CD28 co-stimulation pathway that plays a critical role in priming islet-reactive Th cells 88 Note. RTX = rituximab; RA = rheumatoid arthritis; CD = Cluster of differentiation; (comment: again, CDs are standard abbreviations and much more known than the cluster of differentiation); RANKL = receptor activator of nuclear factor kappa-B ligand; NK = natural killer; IL = interleukin; IFN = interferon; TGF = transforming growth factor; MMP = matrix metalloproteinase; SLE = systemic lupus erythematosus; BAFF = B-cell activating factor; BR3 = BAFF receptor 3; TNF = tumor necrosis factor; ITP = idiopathic thrombocytopenic purpura; MS = multiple sclerosis; CSF = cerebrospinal fluid; DM1 = type 1 diabetes mellitus. Table 2, studies of various autoimmune diseases showed that in addition to B-cell depletion, RTX decreases absolute and/or relative numbers of T cells and T-cell subsets, in particular Th cells. [51][52][53]65,75,76,80,81,83 In addition, a small, likely pathogenic subset of T cells has been identified in patients with RA; they express small amounts of CD20 (CD20 dim ) and are targeted by RTX. These cells are characterized by a shift from Th1 to Th17 subtype, associated with T-cell-mediated inflammation. 89 A fraction of IL-17-producing CD3 + CD4 − CD8 − ("double negative") T cells and CD4 + Th17 cells co-express CD20 on the cell surface and are depleted by RTX in primary Sjogren's syndrome. 90 A similar phenomenon has been demonstrated for patients with MS. 84,91 Reports describing the distribution of B-cell and T-cell subsets in MCD are limited and show variable results. Estevez et al found increased peripheral B-cell counts in 23 children in (untreated) relapse, compared with 26 normal children, whereas the overall number of lymphocytes and total T cells, and CD4 + and CD8 + T-cell counts were unchanged. 92 Others confirmed increased total and relative peripheral CD19 + B-cell counts in treatment-naïve children at the onset of SSNS compared with normal controls and otherwise healthy children with a recent viral infection. 93,94 Lama et al noted a decreased proportion of CD4 + cells both in children with SSNS in relapse and SRNS, 95 whereas Prasad et al showed that Th1 and Th2 cells were increased at the time of relapse and decreased during glucocorticoidinduced remission, measured at least 4 weeks after the discontinuation of prednisone. 96 Significantly greater numbers of CD4 + and CD8 + memory T-cell subsets (CD45RO + CD4 + and CD45RO + CD8 + ) have been described in patients with active, untreated INS compared with healthy controls. 97 According to a model proposed by Lund and Randall, 48 RTX-induced B-cell depletion can interrupt a feed forward loop between autoreactive effector B cells and T cells and abrogate consequent inflammation and tissue injury. Such a mechanism may contribute to the effect of RTX in MCD. It is also possible that RTX works by targeting a small subset of T cells that express CD20. Possible roles of specific T-cell subsets including Treg, Th1, Th2, and Th17 cells in MCD are discussed in the following sections.

Modulation of Treg Cells and Related Cytokines
Thymus-derived, naturally occurring Treg cells are defined by the combined detection of CD4, CD25, and the transcription factor, Forkhead box P3 (Foxp3). 98 Treg cells function as immune suppressors through several mechanisms, including the production of inhibitory cytokines, IL-10, and TGFβ. 99 Expression of Foxp3 in mature Treg cells is necessary for their suppressive function, whereas loss of Foxp3 expression leads to the production of cytokines characteristic of other Th cell lineages. 99 Maintaining the division between regulatory and effector T-cell lineages appears to be critical for immune homeostasis; loss of Treg cells is associated with autoimmunity and, possibly, chronic inflammation. 100 Although data presented by Araya et al suggest that the percentage of Treg cells is similar in healthy controls and pediatric patients with MCD in relapse or in remission, 101 others have reported that the number and percentage of peripheral Treg cells (CD4 + CD25 + Foxp3 + ) were significantly decreased during active MCD, compared with patients in remission and healthy controls. 96,[102][103][104][105] In contrast, the peripheral Th1 and Th2 cell abundance was increased during active nephrotic states and decreased during remission, along with the ex vivo expression of interferon (IFN)-γ and IL-4, major cytokines of Th1 and Th2 cells, respectively. During remission, Treg cell numbers rose, with a corresponding increase in IL-10 and TGF-β production ex vivo. 96,[102][103][104] Similar findings were noted in adult MCD patients; the peripheral blood Th17/Treg cell ratio and Th17-related plasma cytokines (IL-17 and IL-23) were increased and Treg cells and Treg-related cytokines (TGF-β1 and IL-10) were decreased during proteinuria and normalized after the induction of remission. 106 Jaiswal et al found lower percentages of Treg cells and Treg/Th1 ratios in glucocorticoid resistant MCD, compared with glucocorticoid responsive MCD in remission and healthy controls. 96,102-104 Shao et al suggested a role for a dynamic equilibrium between Th17 (effector) and Treg cells in patients with various glomerulopathies. 104 Interestingly, in the Buffalo/Mna rat model of FSGS, Treg cells were increased in drug-induced remission, and transfer of Treg cells induced remission. 107 The finding is consistent with the notion that decreased Treg cell numbers or activity may contribute to the development or exacerbation of nephrotic syndrome.
As shown in Table 2, RTX may increase the number of Treg cells and restore their defective regulatory functions in autoimmune diseases such as SLE and ITP. 66,74 Rocatello et al reported an up to 10-fold increase in Treg cells from baseline in patients with active (nephrotic) membranous nephropathy following RTX-induced B-cell depletion and clinical remission. This effect was inversely correlated to the abundance of active CD8 + T cells. 108 How RTX augments the number and/or function of Treg cells remains unclear. Nonetheless, it is possible that RTX increases or restores functions of Treg cells in MCD, thereby preventing inflammatory cytokine-induced podocyte injury.

Th1/Th2 Balance and Related Cytokines
Th1 and Th2 are 2 subsets of Th cells, each expressing specific markers and representative cytokines. Several studies have addressed potential disturbances of the physiological Th1 and Th2 cell polarity in MCD. Although their findings suggest that Th1/Th2 imbalance may have a contributory or causative role in the pathogenesis of MCD, the origin and significance of the observed imbalance remain inconclusive. 109 Some authors reported increased plasma levels or in vitro expression of the main Th1 cytokines IFN-γ and tumor necrosis factor (TNF)-α in MCD patients 95,96,110 ; one study found that remission of proteinuria correlated with decreased IFN-γ levels. 96 However, others failed to observe an increase in Th1 cytokine levels in MCD patients. [111][112][113][114] Evidence to support a role of Th2 cells in MCD appears somewhat stronger but is not conclusive either. The major Th2 cytokines are IL-4 and IL-13. Increased IL-13 serum levels have been reported in patients with active, relapsing proteinuria, 112,114,115 although decreased IL-4 levels have also been reported during relapse. 113 Polymorphisms of IL-13 and IL-4 encoding genes and modification of their signaling pathways have been associated with predisposition to MCD and response to treatment, [116][117][118] providing additional support for the possible role of Th2 cells. Receptors for IL-13 and IL-4 are expressed in cultured podocytes, and both cytokines have been shown in vitro to modulate protein trafficking, proteolysis, intercellular junction, and membrane functions in podocytes. [119][120][121][122] Lai et al reported significant proteinuria and fusion of podocyte foot processes in IL-13 overexpressing rats, associated with increased glomerular expression of CD80, IL-4R, and IL-13R2 and downregulation of nephrin, podocin, and dystroglycan expression. 123 Although these findings are intriguing, they have not been validated by others. Nonetheless, RTX has been shown to reduce IL-13 levels in patients with atopic eczema, 124 suggesting that the action of RTX in MCD may involve the reduction of IL-13 expression. 125

Modulations of Other T-Cell Subsets and Cytokines
Th17 cells. Liu et al noted a significant increase in Th17 cell numbers as well as plasma levels of Th17-related cytokines, IL-17 and IL-23, in MCD patients. 106 Recombinant murine IL-17 exerts a proapoptotic effect in cultured mouse podocytes. 126 Th17 cells may play a role in the pathogenesis of MCD. RTX treatment was associated with a decrease in Th17 cells and Th17-related cytokines (particularly IL-17) in affected tissues and in the circulation of patients with primary Sjogren's syndrome 127 and RA. 128 It is tempting to speculate that Th17 cells play a role in the pathogenesis of MCD and that the reduction of Th17 cells is one of the actions of RTX in MCD.
Other cytokines and possible permeability factors. Other cytokines have been reported to be increased in biological samples from MCD patients during relapse of proteinuria, many of which decreased during remission, for example, IL-1 and IL-6, 129 IL-2, 95,110,111,130,132 and IL-18. 112,133 However, experimental approaches varied widely among studies; investigators chose limited and variable cytokine sets; biological materials used included serum/plasma, urine, peripheral blood mononuclear cells (PBMCs), or supernatant of PBMCs cultured ex vivo with or without stimulation; cytokines were directly measured or mRNA was quantified. Methodological differences and varying or conflicting results complicate drawing definitive conclusions on the roles of each cytokine. Importantly, observed changes in cytokine expression may be the cause or a consequence of MCD exacerbation. 3 Nevertheless, RTX has been shown to reduce the levels of a number of plasma or locally expressed cytokines in humans with RA. 62 RTX treatment of streptozotocin diabetic rats decreased staining of the proinflammatory nuclear factor, nuclear factor-κB, in the kidney. 134 It is therefore possible that RTX acts by altering the expression of certain cytokine(s) and suppressing subsequent inflammation. Of note, several non-cytokine molecules have been proposed to act as circulating permeability factors in INS, such as hemopexin, CLC-1, and suPAR. 135 Although not explored to date, it is conceivable that expression of such molecules may be modifiable by RTX.

Modulation of T-Cell Functions by Reducing Co-stimulation
B cells modulate T-cell responses via co-stimulatory molecules. 136,137 Co-stimulation can be induced by the interaction of CD40 on B cells with CD40L (CD154) on T cells, and by the interaction of CD80 (B7-1) or CD86 (B7-2) on B cells and other antigen presenting cells with CD28 on T cells. Blockade of CD40-CD40L interaction by antibody has been protective against renal injury in mouse models of chronic proteinuric renal disease, 138,139 membranous nephropathy, 140 and lupus nephritis. 141 In patients with SLE, RTX therapy was associated with a significant reduction in CD40L mRNA and downregulation of CD40L on Th cells as well as downregulation of CD40 and CD80 on remnant B cells. These changes correlated with decreased T-cell activation and a favorable clinical response. [66][67][68] It is conceivable that RTX blocks T-cell-costimulatory pathways in MCD, thereby modulating T-cell function.
Interestingly, CD80 can be expressed by podocytes. 142 Its urinary excretion is elevated in patients with relapsing MCD compared with patients in remission, patients with other glomerular diseases, and healthy controls. [143][144][145][146] Sera from MCD patients in relapse, but not in remission, stimulate CD80 expression in cultured podocytes. 147 Increased CD80 expression alters podocyte actin cytoskeleton and slit diaphragm protein organization independent of T and B cells. 142 Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4, also known as CD152) binds to and blocks CD80. 148,149 Yu et al reported that abatacept (a fusion protein composed of the Fc region of immunoglobulin IgG1 and the extracellular domain of CTLA-4), which is used as an inhibitor of CD80, induced partial or complete remission of proteinuria in patients with primary FSGS or post-transplant recurrent FSGS. The authors suggested that abatacept acts by inhibiting CD80 on podocytes. 150 Although experimental data are convincing, there remains the possibility that abatacept acts by blocking canonical T-cell co-stimulation. Of note, constitutive expression of CTLA-4 by Treg cells is believed to be essential for the immuno-inhibitory functions of these cells. 151 The CTLA-4/CD80 pathway and Treg cell functions in the treatment of MCD warrants further exploration. Another co-stimulatory receptor, CD40, was also found in glomeruli or podocytes, 138,152 and its pathological role was suggested in post-transplant recurrence of FSGS, although the evidence was indirect. 152

Role of Antigen Presentation by B Cells
B cells present antigens to T cells. 153 Considering that relapses of proteinuria are often triggered by non-specific viral infections, it is tempting to hypothesize that RTX depletes B cells carrying the memory of the viral antigens and that the lack of viral antigen presentation to T cells leads to a long-term remission. It has been suggested recently that the delayed reconstitution of switched memory B cells after RTX-induced B-cell depletion was protective against relapse of MCD. 49 Although the physiological significance of these findings is yet to be understood, it suggests that memory B cells are important in the pathogenesis of MCD relapse.

Effect on Cytotoxic Cells and Macrophages
Some studies suggested that active MCD is associated with an increased number of cytotoxic cells as evidenced by a decreased CD4 + /CD8 + ratio. 95,113 However, data on a possible role of CD8 + T cells in MCD are scarce. Natural killer (NK) cell deficiency was associated with frequently replacing MCD in a patient with Hodgkin's disease, 154 and RTX was shown to increase active NK cells in patients with RA and SLE, 59,60,70 suggesting a possible implication of NK cells in the effect of RTX in MCD. However, another study reported functionally normal NK cells in MCD. 92 RTX was also shown to decrease the number of synovial macrophages (Mϕ) 53 and to inhibit TNF-α production by Mϕ in patients with RA. 57 Although the data suggest a role for TNF-α in some patients with recurrent FSGS post-transplantation, 155 the role of TNF-α and/or Mϕ is unknown for MCD. With currently available evidence, there is no strong signal to implicate cytotoxic cells, NK cells, or Mϕ in the mechanisms of action of RTX in MCD, and it appears that the field is understudied.

Direct, Non-immunological Effects on Podocytes
Although podocytes do not express CD20, Fornoni et al reported that RTX binds to podocytes by targeting sphingomyelin-phosphodiesterase-acid-like 3b (SMPDL-3b), which regulates acid-sphingomyelinase (ASMase) activity. 50 SMPDL-3b and ASMase are essential for the organization of receptors and signaling molecules in highly specialized cells 156 and are involved in the modulation of actin remodeling in podocytes. 50 In Fornoni's study, RTX partially prevented the downregulation of SMPDL-3b and ASMase in cultured podocytes exposed to FSGS serum; SMPDL-3b overexpression or treatment with RTX prevented disruption of the actin cytoskeleton and podocyte apoptosis induced by patients' sera. This effect was diminished when the SMPDL-3b gene was silenced. 50 Yoo et al suggested that SMPDL-3b may be an important modulator of podocyte function and a novel therapeutic target in glomerular diseases. 157 Thus, RTX may improve SMPDL-3b activity in podocytes and contribute to its therapeutic effect in MCD.

Conclusions and Future Research Direction
The aim of this review was to synthesize hypotheses pertaining to the mechanisms of action of RTX in the induction of prolonged remission in MCD. Insights into these mechanisms can be exploited to study the pathogenesis of MCD; understanding how RTX works in the treatment of MCD is expected to shed new light on the immune pathogenesis of the disease and will possibly lead the identification of the elusive permeability factor(s).
Although the direct protective actions of RTX on podocytes remains a possibility, overwhelming evidence points the involvement of the immune system, in particular T and B lymphocytes. Although the direct actions of lymphocytes on podocytes are yet to be examined, evidence points to the presence of circulating humoral factors responsible for podocyte injury in MCD, whose identity is yet to be uncovered. 135 We would like to propose a hypothesis that in MCD, RTX targets disease-promoting B-cell subsets that promote dysregulated T-cell responses, which are responsible for the secretion of humoral factors that injure podocytes. Alternative hypotheses would be that RTX targets a small number of inflammatory CD20 + T cells 91 or effector B cells. 135 Recent advances in high-resolution immunophenotyping technologies allow the robust enumeration, stratification, and functional characterization of immune cell subsets including T-cell and B-cell subsets, which we can use to pin-point the immune derangement in MCD. 158 With the increasing number of MCD patients treated with RTX for relapse control, we now have tools and opportunities to test our hypotheses. Efforts toward such investigations are underway in our laboratory in collaboration with the Canadian Childhood Nephrotic Syndrome Project. 159

Author Contributions
NM performed literature review and drafted the manuscript. MB contributed to literature review and writing of the manuscript. TT supervised the entire process of manuscript preparation. All authors read and approved the final manuscript.

Ethics Approval and Consent to Participate
Not applicable.

Consent for Publication
All authors have consented for article publication.

Availability of Data and Materials
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Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study is supported by grants from the Canadian Institute of Health Research (MOP-53335, MOP-126180 to TT) and the Kidney Foundation of Canada (to TT).