The current evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome : Critical review

Introduction Rituximab is a chimeric monoclonal antibody directed against the CD20 receptor on B cells present along all maturational stages of its cycle but not in plasma cells. The administration of one dose of 375 mg/m2 per week for 4 weeks or the administration of a total dose 2 g within a 2-week interval causes depletion of circulating B lymphocytes, which lasts for 6–7 months. Although rituximab was designed for the treatment of relapsing or refractory non-Hodgkin lymphoma B, since its introduction in the therapeutic scene, it was considered an attractive drug for the treatment of various autoimmune diseases mediated by antibodies. The aim of this critical review was to assess the evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome. Conclusion Current evidence from observational studies suggests that rituximab can induce the remission of nephrotic syndrome in patients suffering from membranous glomerulonephritis, minimal change nephropathy and focal segmental glomerulosclerosis. However, there is an absence of controlled clinical trials to compare rituximab against the standard of reference in each case, which should be used to define specific indications. With the level of current evidence, it seems reasonable to consider that rituximab is a therapeutic option for patients who have contraindications, intolerance or lack of response to conventional treatment regimens and for patients who have demonstrated dependence of calcineurin inhibitors and are exposed at the risk of chronic nephrotoxicity. Introduction Rituximab is a chimeric monoclonal antibody (murine/human) (Figure 1), designed to bind specifically to the CD20 receptor—lymphocyte differentiation antigen B—present in the cell membrane of pre-B cells and mature B but not in the plasma cells. The binding of rituximab to CD20 causes a depletion of B lymphocytes through three mechanisms * Corresponding author Email: alsegarr@gmail.com Servicio de Nefrologia, Hospital Vall d’Hebron, Barcelona, Spain Figure 1: Rituximab is a chimeric monoclonal antibody with a human constant domain (IgG1), a human kappa constant domain and a murine variable region through which it binds to its specific receptor CD20, located on the surface of B lymphocytes. —antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis1,2 (Figure 2). The depletion of circulating B lymphocytes may have implications in the normal immune response both for their role as precursors of antibody-producing plasma cells and for their recognized role as antigen-presenting cells. Although rituximab was conceived and registered for the treatment of refractory or relapsing B non-Hodgkin lymphoma, since its introduction into the therapeutic scene, it was considered an attractive drug for treating various autoimmune diseases mediated by antibodies. We critically review the current clinical experience sup-porting the treatment with rituximab in primary glomerulopathies causing nephrotic syndrome.


Introduction
Rituximab is a chimeric monoclonal antibody (murine/human) (Figure 1), designed to bind specifically to the CD20 receptor-lymphocyte differentiation antigen B-present in the cell membrane of pre-B cells and mature B but not in the plasma cells.The binding of rituximab to CD20 causes a depletion of B lymphocytes through three mechanisms Figure 1: Rituximab is a chimeric monoclonal antibody with a human constant domain (IgG1), a human kappa constant domain and a murine variable region through which it binds to its specific receptor CD20, located on the surface of B lymphocytes.
-antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis 1,2 (Figure 2).The depletion of circulating B lymphocytes may have implications in the normal immune response both for their role as precursors of antibody-producing plasma cells and for their recognized role as antigen-presenting cells.Although rituximab was conceived and registered for the treatment of refractory or relapsing B non-Hodgkin lymphoma, since its introduction into the therapeutic scene, it was considered an attractive drug for treating various autoimmune diseases mediated by antibodies.We critically review the current clinical experience sup-porting the treatment with rituximab in primary glomerulopathies causing nephrotic syndrome.

Rituximab as a first-line therapy
The first evidence on the efficacy of rituximab in MN was reported by Remuzzi et al. 7 in a group of eight patients with primary MN and longstanding nephrotic syndrome.Following the administration of one dose of 375 mg/m 2 rituximab weekly for four consecutive weeks, there was a reduction in proteinuria.After a 20-week follow-up, the percentage of patients in complete or partial remission of nephrotic syndrome (62.5%) was only slightly lower than that reported with alkylating agents and calcineurin inhibitors (70-80%).The reduction of proteinuria was associated with depletion of circulating B cells, which was evident after the first dose and maintained throughout the observation period of 20 weeks.In a second article 8 , the same group described the 12-month follow-up of eight patients treated with the same regimen.The reduction in proteinuria was quantitatively higher in the first 3 months after treatment and persisted throughout the study period with a percentage of total or partial remissions of 50% at 12 months.These studies provided the first clinical data on the efficacy of rituximab in MN but also showed that not all patients respond to treatment.In 2006, Ruggenenti et al. 9 analysed the possible predictors of response  3,4 .Inhibi-tion of B-cell function reduces pro-teinuria in experimental models of several MN, and in the same line 5 , clinical studies 6

Rituximab in membranous glomerulonephritis
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.
21 months after treatment, there was a significant increase in fractional sodium clearance, a decrease of renal plasma flow and an increase in renal vascular resistance.Repeated biopsies showed significant reduction or disappearance of subepithelial deposits in association with a reduction in IgG4 deposition and with an increase in the number of electrondense filtration diaphragms.Overall, they concluded that the morphofunctional changes observed after the disappearance of proteinuria could have a renoprotective meaning (Table 1a).

Rituximab associated to calcineurin inhibitors
The likelihood of remission in patients with MN treated with calcineurin inhibitors depends on the total duration of treatment.follow-up, 4 were in complete remission, 12 were in partial, 1 had a limited response and 1 patient relapsed.
Similar observations were reported by Cravedi et al. 18 in a matched cohort study comparing 2-year outcomes of 11 consecutive patients with primary MN who received rituximab for persisting nephrotic syndrome or relapsing disease.Finally, a recent observational study, which included 100 patients with MN treated with rituximab, described a frequency of partial or total remissions of 65%, whether rituximab was indicated as first-line treatment or is administered after prior exposure to other immunosuppressants 19 .
In summary, the current data, albeit with low level of evidence, are homogeneous and indicate that rituximab may have a role in the treatment of membranous glomerulonephritis.However, there is not enough data to define the indications against alkylating agents or calcineurin inhibitors.A multicentre randomized control study comparing the use of rituximab versus cyclosporine in the treatment of MN has recently been finished and will provide valuable information on this subject.Moreover, despite the growing demonstration of positive responses to rituximab in MN, the evidence that this disease tends to have a chronic clinical course with intercurrent outbreaks of activity raises doubts about the ability of this drug to modify the long-term clinical course, especially taking into account that the re-exposure to rituximab can induce the formation of neutralizing antibodies that limit their effectiveness.For the of patients that remain in remission with no treatment at 12 and 18 months.Though promising, these data should be the subject of rigorous evaluation in randomized studies before attempting to move them to clinical practice.A second study 16 provided new data demonstrating the utility of rituximab to overcome the dependence of calcineurin inhibitors.This study included adult patients with idiopathic membranous GM who were in full or partial remission but had a long history of relapses and clear criteria for dependence on calcineurin inhibitors.Following administration of one cycle of four doses of rituximab, it was possible to remove the calcineurin inhibitors in all cases.During follow-up, the recovery of B cells was not associated with recurrence of nephrotic syndrome.Three patients had late recurrences long time after administration of the first treatment cycle, and in all of them, remission was achieved with the administration of a single dose of 1 g of rituximab.After extended follow-up, all patients remained in remission without calcineurin inhibitors (Table 1b).

Rituximab in patients resistant to standard therapies
There is also limited, low-level evidence indicating that rituximab could be useful in patients with MN resistant to conventional therapies.Fervenza et al. 17 have recently extended the experience with rituximab in a study including 20 patients (11 of which had failures to prior therapy).All patients received retreatment at 6 months regardless of proteinuria response.Of 18 patients who completed 24 months be very long.Two pilot studies have analysed whether the use of rituximab in combination with calcineurin inhibitors can shorten the treatment period required to obtain remission and/or obtain stable remissions of proteinuria after the withdrawal of calcineurin inhibitors.Regarding the first aspect, a recent controlled nonrandomized clinical trial 15 analysed whether the association of rituximab with tacrolimus could induce earlier and more stable remissions of proteinuria than monotherapy with tacrolimus in patients with primary MN.The study included 18 consecutive patients with primary MN and normal renal function with nephrotic syndrome lasting for more than 6 months despite treatment with angiotensin II blockers.All patients started treatment with tacrolimus monotherapy at doses of 0.06 mg/ kg/day, which was subsequently adjusted to maintain through levels within 7-9 ng/ml.During the first 6 months after tacrolimus, all patients were treated with one dose of 375 mg/m 2 of rituximab/week for four consecutive weeks.After administration of the last dose of rituximab, tacrolimus dose was reduced by 30% per month until either discontinuation of treatment or evidence of relapse.The probability of response at each time period was compared with a cohort of 36 patients treated with tacrolimus monotherapy or in association with steroids.The results of this study indicate that the association of tacrolimus to rituximab is associated with a significantly lower proteinuria at 9 months after initiation of treatment, can prevent recurrence after suppressing calcineurin inhibitors and increases the number

Rituximab in primary focal and segmental glomerulosclerosis (FSGS)
The rationale to indicate rituximab treatment in FSGS is based on the hypothesis that the podocyte injury is caused by an inmunomodulable, B-cell-related, pathogenic mechanism 39 .However, recent evidence indicates that rituximab could reduce proteinuria for having direct effects on intracellular signalling pathways and podocyte cytoskeleton architecture 40 .There are isolated case reports and observational studies on the efficacy of rituximab in patients with FSGS [41][42][43][44][45][46][47][48] .The results are discordant as regards to the effect both on proteinuria and on renal function.Thus, the available evidence is not enough to ascertain whether rituximab should or should not be indicated in patients with steroid-resistant FSGS.The results of individual case reports suggest that there might be a better response to rituximab in children than in adults, but this point alteration seen is the effacement of podocyte foot processes seen by the electron microscope.The cause (or causes) of podocyte injury has been the subject of numerous studies and is today still a matter of debate 20 .The favourable response of nephrotic syndrome to immunosuppressant agents has been considered as an undeniable proof of the involvement of the immune system.Most of the evidences indicate an important role of T cells in the pathogenesis of MCN 20,21 .However, the report of an unexpected resolution of the nephrotic syndrome in a patient treated with rituximab for a B-cell lymphoproliferative disorder 22 led to rethink the role of B cells in the pathogenesis of MCN.Since then, several case reports and small series of patients have confirmed the evidence of remission of nephrotic syndrome with rituximab in both adults and child patients with minimal change disease (MCD).So far, the evidence is limited to cases in which the indication has been steroid resistance, the occurrence of multiple relapses, dependence or intolerance to classic drugs such as corticosteroids and calcineurin inhibitors.The reported evidence [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] is summarized in Tables 2a and 2b.
In patients with steroid and/or CNI dependence, rituximab can reduce the number of relapses and induce sustained remissions in about 70% of cases, but 25% of patients will require retreatment to maintain remission.The available data for steroid-resistant patients are scarce and limited to case reports 27,28,32 and an uncontrolled study including 16 patients 54 .Overall, the results indicate that about half of the patients may time being, it seems rational to consider treatment with rituximab in patients who do not respond or have contraindications to conventional treatment regimens and in patients who have developed dependence to calcineurin inhibitors.The mechanisms by which rituximab can induce remission of nephrotic syndrome in MN are not known.It seems logical to assume that its main effect should be explained by a reduction of the synthesis of antibodies and/or the interference with the function of Blymphocyte and antigen-presenting cell, secondary to depletion of circulating B cells; however, there is some evidence that the explanation would not be so simple.First, after treatment with rituximab, B-cell depletion occurs in all patients while only 60-70% of them show total or partial response 7,8,11 .Second, recovery of circulating B cells is not associated with a recurrence of nephrotic syndrome 8,9,11,16 .Finally, in the studies reported by Remuzzi et al. 7 and Ruggenenti et al. 8 , although the maximum effect on proteinuria was seen within the first 1-3 months after treatment, proteinuria continued to decline after the sixth month when, in some patients, there was a recovery in the number of circulating CD19 cells (Table 1c).All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

Rituximab in minimal change
has not been adequately analysed.
Although the results are also discordant, treatment with rituximab, usually associated with plasmapheresis, has been associated with greater apparent success in treating the posttransplant recurrence of FSGS when compared with FSGS of the native kidney 44,45 .However, the data observed in post-transplant recurrence cannot be extrapolated to primary FGS of native kidney for several reasons.First, since recurrence is only seen in approximately 30% of patients, it could be associated with a particular pathogenic mechanism, which may be different than those causing podocyte injury in nonrecurrent forms.Second, in transplant patients, relapse is evident in a background of combined strong immunosuppression, so the mechanisms responsible for recurrence appear to be quite insensitive to immunomodulation.Finally, post-transplant recurrence is usually diagnosed by the evidence of proteinuria, which in several cases could even not reach the nephrotic range [44][45][46] .Therefore, it is possible that the treatment of relapsing forms is initiated in earlier stages of podocyte injury and therefore most likely to respond to therapeutical measures (Table 3).

Rituximab for steroid-dependent idiopathic nephrotic syndrome
Recent studies [47][48][49][50][51][52][53][54] analysed the effectiveness of rituximab in patients with steroid-dependent idiopathic nephrotic syndrome. These tudies, however, included patients with different renal diseases (MCD, FSGS, mesangial hypercellullarity and patients in whom biopsy data were not available), and in all cases, results are described for the whole group but not detailed for each particular renal disease.This is the reason why these studies are described separately.The results of observational studies [49][50][51][52][53][54] are similar to those described in patients with steroid-dependent MCN and are summarized in Table 4. Overall, data from these studies indicate that approximately 80% of patients with steroid dependence can maintain remission after treatment with rituximab.Most patients, however, suffer one or more relapses and need to be retreated.In the long term, 40-50% of patients can maintain remission with no other immunosuppressive therapy.The efficacy of rituximab versus other drugs in preventing relapses in patients with steroid dependence has been investigated in two studies-an observational trial 50 and a randomized clinical trial 53 .
In the first one 50 , the number of relapses observed in patients treated with three to four repeated doses of rituximab was comparable with that observed in a control group of patients treated with tacrolimus for 12 months 50     * One patient with FSGS and 11 patients with MCD.Steroids were discontinued in all cases after 74 days.Frequency of relapses per 6 months was significantly reduced and steroid-free period per 6 months was significantly increased after treatment compared with those before treatment.† Four patients with MCD and two with FSGS and four patients with no biopsy data available.The study compared the frequency of relapses after Rtx or TCL for patients with difficult-to treat steroid-dependent nephrotic syndrome and showed that two or three doses of Rtx were as effective as 12 months treatment with TCL to reduce the frequency of relapses and had an steroid-sparing effect.‡ Ten patients with MCD, 10 patients with FSGS and 2 patients with no biopsy data.Remission without steroids and calcineurin inhibitors: n, 20.Treatment failure, two patients.¶ No biopsy data available.After the initial rituximab course for >24 months, 7 (24.1%)patients without further maintenance immunosuppression.After repeated rituximab doses, 69% of patients remained on remission and 48% free of associated immunosuppression.§ Twelve patients with MCD, two patients with FSGS two patients mesangial hypercellularity, eight patients with no biopsy.Remission was sustained in 20 (83.3%) at 12 months and in 17 (71%) at follow-up of 16.8 ± 5.9 months.The mean difference in relapses before and 12 months after treatment with rituximab was 3.9 episodes/patient per year.MCD, minimal change disease; FSGS, focal and segmental glomerulosclerosis; MMF, mycophenolate; MZR, mizoribine; TCL, tacrolimus; CyA, cyclosporine A.

Figure 2 :
Figure 2: Upon binding to its receptor, rituximab induces B-cell depletion through three different mechanisms: (a) antibody-dependent cytotoxicity, (b) complement-mediated cell lysis and (c) induction of apoptosis.

Table 1 Rituximab in idiopathic membranous nephropathy 1a Rituximab as a first-line therapy Reference
essary re-exposure to the drug while reducing the cost of treatment.One area of great interest is the pharmacokinetics of rituximab in nephrotic syndrome.Since rituximab is a 231 amino acid phosphoprotein with a molecular weight of 145 Kd, urinary losses of rituximab could limit its n Response (%) Total remission (%) Partial remission (%) No response (%) Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) For citation purposes: Segarra Medrano A, Romero Jaller K.The current evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome: Critical review.OA Nephrology 2013 Apr 01;1(1):3.Competing interests: none declared.Conflict of interests: none declared.All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

1b Rituximab associated to calcineurin inhibitors
complete or partial remission after treatment with rituximab but relapse occurs in 50% of them.The absence of clinical trials comparing the efficacy of rituximab against other therapeutic options prevents conclusions about what should be its specific indications.As in the case of MN, rituximab should be considered as a therapeutic option for patients with multiple recurrences that develop dependence to calcineurin inhibitors and for difficult-to-treat patients who are resistant to firstand second-line treatments (steroids, calcineurin inhibitors and alkylating agents).
Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY)For citation purposes: Segarra Medrano A, Romero Jaller K.The current evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome: Critical review.OA Nephrology 2013 Apr 01;1(1):3.Competing interests: none declared.Conflict of interests: none declared.All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.have

Table 2 Rituximab in minimal change nephropathy 2a Rituximab in steroid dependence and multiple recurrences Reference n Indication Associated treatment Rtx dose Response Relapse Retreatment Follow-up months
CR, complete remission; CsA cyclosporine; Ig, high-dose immunoglobulins, MZR, mizoribine; MMF, mycophenolate mofetil.Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY)For citation purposes: Segarra Medrano A, Romero Jaller K.The current evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome: Critical review.OA Nephrology 2013 Apr 01;1(1):3.Competing interests: none declared.Conflict of interests: none declared.

Rituximab in steroid-resistant patients Reference n Indication Associated treatment Rtx dose Response Relapse Retreatment Follow-up months
Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY)For citation purposes: Segarra Medrano A, Romero Jaller K.The current evidence supporting rituximab treatment in primary glomerular diseases causing nephrotic syndrome: Critical review.OA Nephrology 2013 Apr 01;1(1):3.Competing interests: none declared.Conflict of interests: none declared.All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.