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The efficacy and dynamic changes of immune function of rituximab with mycophenolate mofetil in the treatment of steroid-dependent /frequently relapsing nephrotic syndrome: a retrospective follow-up study

BMC Nephrology volume 26, Article number: 186 (2025) Cite this article

Abstract

Introduction

Approximately 70%～90% of children with steroid-sensitive nephrotic syndrome (SSNS) will suffer from steroid dependency or frequent relapses, prompting the use of steroid-sparing agent. In this study, we investigate the efficacy and the characteristics of dynamic changes in immune function of two doses of rituximab (RTX) in the treatment of steroid-dependent/frequently relapsing nephrotic syndrome (SDNS /FRNS).

Method

Retrospective follow-up study was conducted in our hospital from June 2022 to September 2023. 7 children with SDNS /FRNS were allocated to intravenous 2 doses RTX (each dose 375mg/m2, 1 dose per week) and administered the standard oral dose of mycophenolate mofetil (MMF) (1000–1200/m2/d, divided into 2 doses) when B cells have recovered (≥ 5/ul). The study subjects after treatment were monitored for the efficacy and dynamic changes of immune function for 12 months.

Result

7 children with SDNS/FRNS who were treated RTX with MMF and followed up for 12 months have no relapse. The rate of B cell depletion (< 5/ul) was 100% at 1 week after the second dose of RTX treatment, and the rate of B cell recovery was 100% at 5–12 months after the first dose of RTX treatment. There was no significant difference with T cell subsets (CD3, CD4, CD8, CD4/CD8) at each follow-up time points (all P > 0.05). The count of NK cells was significantly higher than that of other groups at 1 week after the second dose (P < 0.05). The IgM level at 1 week after the second dose was significantly lower than that before treatment and 1 week after the first dose (P < 0.05). There were no significant differences with IgA, IgG, C3 and C4 before treatment, 1 week after the first dose and 1 week after the second dose (all P > 0.05).

Conclusion and recommendation

Administering two doses of RTX along with the standard dose of MMF has been effective in maintaining remission for children with SDNS/FRNS. B cell depletion can be achieved one week after the second dose of RTX treatment. NK cell proliferation may play a role in B cell depletion, and early B cell depletion may suppress the production of IgM. These findings require further validation through additional clinical trials and basic research.

Peer Review reports

Introduction

Idiopathic nephrotic syndrome (INS) is the most frequent glomerular disease in children, characterized by massive proteinuria, hypoalbuminemia (serum albumin < 25 g/L), hyperlipidemia, and/or varying degrees of edema [1]. Approximately 70- 90% of children with INS respond to glucocorticoid therapy but are susceptible to relapses. Over 50% of children with SSNS will experience FRNS, some may develop SDNS, and 10–20% are resistant to steroid treatment [2]. Glucocorticoid therapy is the foundation of INS treatment, and for those with SDNS or FRNS, steroid-sparing agents such as cyclophosphamide, tacrolimus, or MMF can be added to the treatment [3]. Furthermore, the 2020 IPNA guidelines recommend the use of calcineurin inhibitors as first-line induction therapy for steroid-resistant nephrotic syndrome (SRNS) [4]. However, at least 50% of children with SSNS who are treated with steroid-sparing agents experience frequent relapses or become dependent on steroids, and 15% of children with SRNS show resistance to these agents in clinical practice [2,3,4].

In recent years, RTX has shown promising efficacy in the treatment of SDNS, FRNS, or SRNS [4,5,6]. RTX is a chimeric human-mouse monoclonal antibody that targets the CD20 molecule on the surface of B cells, induce B cell apoptosis and play a significant role in the treatment of INS [7, 8]. Studies have indicated that B cells can produce specific autoreactive antibodies attacking podocytes and inducing proteinuria [9]. Depletion of B cells through RTX treatment may reduce the production of pathogenic antibodies, alleviate podocyte destruction, and thereby decrease proteinuria [9]. Iijima K et al. pointed out that administering MMF when B cells have recovered can fully prevent treatment failure following RTX therapy [10]. Furthermore, B cells, which have antigen-presenting capabilities and can provide costimulatory signals to T cells, may lead to the effacement of podocyte foot processes upon activation. Depletion of B cells through RTX treatment may suppress T cell activation, subsequently reducing the destruction of podocyte foot processes and thereby achieving a therapeutic effect [11]. Currently, the immune mechanisms of RTX in the treatment of INS are not well understood, and there is a paucity of research in this area, necessitating further clinical studies [12, 13]. Most of the studies on RTX treatment for INS still focus on evaluating its efficacy now, and more attempts are needed to explore its immune mechanisms. Therefore, to further understand the dynamic changes in immune function during RTX treatment for INS and to enhance our comprehension of its immune mechanisms, we conducted a follow-up study of clinical and immunological data from 7 children with INS who were treated with RTX and MMF at our hospital from June 2022 to September 2023. We analyzed both the efficacy and the dynamic changes in immune function.

Methods

Population and study design (Fig. 1)

Following ethics board approval (application number: KY-2024-114-K01, Date: 10 September 2024), the study was conducted at the Women and Children’s Hospital, School of Medicine, Xiamen University from June 2022 to September 2023. The hospital is one of the largest hospital in Xiamen, the largest city in southeast Fujian, China. The diagnostic criteria for SDNS/FRNS [2]: INS: 1.24-hour urine protein quantification ≥ 50 mg/Kg or morning urine protein/creatinine (mg/mg) ≥ 2.0 or three consecutive mornings within one week of morning urine protein qualitative tests showing (+++) to (++++). 2. Serum albumin level < 25 g/L. 3. Serum cholesterol level > 5.7mmol/L. 4. Varying degrees of edema. SDNS: Sensitive to steroids, but relapses within two weeks of consecutive dosage reduction or discontinuation. FRNS: Recurrence at least twice within six months or ≥ 4 times within one year during the course of the disease.

Fig. 1
figure 1

Flowchart of study population of SDNS/FRNS with two doses of rituximab (375mg/m2 per dose, one dose per week) and administered the standard oral dose of MMF (1000–1200/m2/d, divided into 2 doses) (N = 7) followed up in our center. Note 1 SDNS: steroid-dependent nephrotic syndrome, FRNS: frequently relapsing nephrotic syndrome, B: B cells, NK: NK cells, CD3: CD3 T-cells, CD4: CD4 T-cells, CD8: CD8 T-cells, CD4/CD8: CD4 T-cells/ CD8 T-cells

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The inclusion criteria for the study subjects were as follows: (1) Age ≤ 18years, no gender restrictions. (2) A confirmed diagnosis of SDNS/FRNS. (3) Completion of two doses of RTX treatment (375mg/m2 per dose, one dose per week) and administered the standard oral dose of MMF (1000–1200/m2/d, divided into 2 doses) when B cells have recovered (≥ 5/ul). The exclusion criteria were: 1. Children with Other (such as oral tacrolimus, etc.) treatment plans. 2.Children who did not complete two doses of RTX (375mg/m2 per dose, one dose per week) or the standard oral dose of MMF (1000–1200/m2/d, divided into 2 doses) when B cells have recovered (≥ 5/ul). 3. Children with incomplete clinical follow-up data or those lost to follow-up.

A retrospective follow-up study was conducted at our hospital. 7 children with SDNS /FRNS were enrolled and followed up for 12 months. The follow-up time points were as follows: before treatment, 1 week after the first dose, 1 week after the second dose, 2 months after the first dose, 3 months after the first dose, 5 months after the first dose, and 12 months after the first dose.

Data collection technique and quality assurance

Thirty minutes prior to the initiation of the RTX infusion, the following premedication were required: 1. A single oral dose of acetaminophen (15 mg /kg). 2. Antihistamines: A single oral dose of desloratadine dry suspension (1.25 mg for children aged 1–5, or 2.5 mg for children aged above 6). 3. Corticosteroids: A single intravenous dose of methylprednisolone (0.8-1.6 mg/kg).

RTX Injection Storage and Administration: The RTX injection should be stored in its original vial in the dark at a temperature range of 2–8 °C. Once prepared, the solution can be stored at room temperature for up to 12 h, or in a refrigerator at 2–8 °C for up to 24 h. The required dose of RTX should be aseptically withdrawn and diluted in a sterile, pyrogen-free 0.9% saline or 5% glucose solution to a final concentration of 1 mg/ml of RTX. The initial infusion rate should be 50 ml/h, maintained for the first hour, and then increased by 25 ml/h every 30 min, not exceeding a maximum infusion rate of 5 ml/kg·h. During the intravenous infusion, continuous monitoring of blood pressure, heart rate, and transcutaneous oxygen levels is mandatory.

Cellular and Humoral Immunity Testing: The BD FACSCantoâ„¢ II flow cytometer was utilized to assess the B cells, NK cells, CD3 T-cells, CD4 T-cells, CD8 T-cells, and CD4 T-cells/CD8 T-cells of each follow-up time point. Additionally, the Siemens BNII full-automatic specific protein detection system was employed to measure IgM, IgA, IgG, C3, and C4 levels of each follow-up times point.

Data Collection: Clinical data for the enrolled children were collected throughout the follow-up period, including details such as gender, age, the process of tapering off or discontinuing steroids, the occurrence of relapses in SDNS/FRNS, and side effects following first dose of RTX treatment, etc. Immunological parameters such as B cells, NK cells, CD3 T-cells, CD4 T-cells, CD8 T-cells, CD4 T-cell /CD8 T-cell, IgM, IgA, IgG, C3, C4 levels were monitored at each follow-up time point. Subsequently, a designated individual organized the data on a computer (Fig. 1).

Statistical analysis

All calculations were performed using commercially available statistical software (IBM® SPSS® Statistics version 22). For normally distributed quantitative data were represented as mean ± S and non-normally distributed quantitative data were represented as medians (interquartile ranges). For statistical analysis to calculate the significance level among multiple groups, values were compared by one-way ANOVA or the nonparametric Kruskal-Wallis test, as appropriate. A difference was considered statistically significant at P < 0.05.

Results

Gender, age, and diagnostic information (Table 1)

A total of 7 subjects fulfilled the inclusion criteria, comprising of 5 males and 2 females, with ages from 5 to 16 years and a median age of 7 years. The disease duration for these 7 cases varied from 1 to 9 years, with a median of 5 years. Among them, 4 cases were steroid-sensitive, 2 were steroid-dependent, and 1 was steroid-resistant. All 7 cases were frequently relapsing, and the last relapse occurred between 7 and 103 months after diagnosis, from 24 to 76 days prior to the commencement of treatment. Renal biopsies revealed 1 case of focal segmental glomerulosclerosis and 1 case of minimal change disease, and 5 cases did not undergo biopsy. Genetic testing in the 7 cases identified 1 case with a c.2984 C > T variation in the FN1 gene (clinical significance unknown) and a c.1314 C > G variation in the PTPRO gene (clinical significance unknown), as well as 1 case with a heterozygous mutation in the NPHS1 gene (clinical significance unknown). Three children received rituximab when they were first diagnosed with SDNS/FRNS. If children had previously transitioned between FRNS and SDNS, some of them had been treated with other immunosuppressive drugs (CTX, MMF, FK506 or Levamisole).

Table 1 Clinical characteristics and efficacy of RTX treatment in children with SDNS/FRNS
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Efficacy and side effects of RTX treatment for SDNS/FRNS (Table 1)

All 7 enrolled children received RTX treatment of two doses (375mg/m2 per dose, one dose per week). Following the first dose, all children were prescribed compound sulfamethoxazole orally (trimethoprim 3 mg/Kg qod) to prevent Pneumocystis jiroveci infection. All 7 children were on full-dose steroids before the start of treatment, but the dosage of steroids had been reduced in some children because of alleviating side effects. If they were taking a daily dose of glucocorticoids, which was changed to every other day after the first dose and gradually tapered (5-10 mg every 2 weeks), with all discontinuing the medication after 4–6 months of treatment. Among the 7 cases, 1 child discontinued medication after 4 months, 4 after 5 months, and 2 after 6 months. All 7 children added MMF (1000–1200/m2/d, divided into 2 doses orally) when B cells have recovered (≥ 5/ul) after two doses of RTX, with the addition time ranging from 5 to 12 months, and a median of 7 months.

All 7 children were followed up for 12 months, with no edema at any of followed-up time points. Over the period of 12-months follow-up, 1 child had urinary protein of 1 + before treatment and again 1 + one week after the first dose, which turned negative one week after the second dose. Another child had urinary protein of 1 + at the 12 -month mark after the first dose, which turned negative spontaneously after 3 days. The remaining 5 children tested negative for urinary protein throughout the follow-up period. During the 12-months follow-up, urinary protein/urine creatinine ratios was negative in all 7 children (urinary protein/urine creatinine < 0.2 mg/mg). Over the same 12-month period, serum albumin levels were normal in all 7 children (serum albumin > 25 g/L). Regarding serum cholesterol, one child had a mildly elevated level, while the other 6 children had approximately normal levels (the average of serum cholesterol < 5.7mmol/L). In summary, none of the 7 children experienced a relapse within the 12 months of follow-up.

All enrolled children underwent RTX treatment with two doses (375mg/m2 per dose, one dose per week), and acetaminophen, antihistamines, and corticosteroids were used 30 min before treatment to prevent non-specific inflammation or allergic reactions, with no fever or rash occurring. During the 12-month follow-up period, all 7 children experienced infections, which were exclusively upper respiratory tract infections. These infections occurred between the administration of the second dose and 6 months after the first dose. Among the 7 children with upper respiratory tract infections, 4 had COVID-19 infections. And, 4 children were infected once (2 with COVID-19 and 2 with an unknown pathogen), 1 child was infected twice (once with COVID-19 and other with an unknown pathogen), 1 child was infected three times (once with COVID-19 and twice with an unknown pathogen), and 1 child was infected four times (all with an unknown pathogen).

Dynamic changes in immune function after RTX treatment for SDNS/FRNS (Table 2)

After the first dose of treatment in the enrolled children, the rate of B cell depletion (B cell count < 5 cells/ul) was 71.4% (5/7), and after two doses of treatment, the rate of B cell depletion was 100% (7/7). The rate of B cell recovery (B cell count ≥ 5 cells/ul) was 57.1% (4/7) within 5 months after the first dose, and the rate of B cell recovery within 12 months after the first dose was 100% (7/7), but in 1 case, the B cell counts was 11 cells/ul. The percentage of NK cells increased 1 week after the second dose in children with SDNS/FRNS treated with RTX, decreased within 2 months after the first dose and 3 months after the first dose, and was mildly elevated 5 months after the first dose (Fig. 2). The B cell count was lower after the first dose and after two doses of treatment compared to before treatment and 12 months after the first dose. Specifically, the count after two doses was lower than after the first dose, and the count 12 months after the first dose was lower than before treatment, with all differences being statistically significant (P < 0.05). There were no significant differences in the counts of CD3 T-cells, CD4 T-cells, CD8 T-cells, and the CD4 T-cell/CD8 T-cell ratio between the following time points: before treatment, after the first dose, after the second dose, 2 months after the first dose, 3 months after the first dose, 5 months after the first dose, and 12 months after the first dose. These differences were not statistically significant (P > 0.05) (Fig. 3). In contrast, The NK cell count was higher after the second dose and 12 months after the first dose compared to before treatment and after the first dose, with these differences being statistically significant (P < 0.05) (Fig. 3).

Table 2 Dynamic changes in immune function in children with SDNS/FRNS treated with RTX
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Fig. 2
figure 2

The transition of the percentage of NK cells and IgM levels for each patient over time. A The percentage of NK cell change in children with SDNS/FRNS treated with RTX, B The percentage stacked line graph of NK cell change in children with SDNS/FRNS treated with RTX, C The percentage of IgM change in children with SDNS/FRNS treated with RTX, D The percentage stacked line graph of IgM change in children with SDNS/FRNS treated with RTX. Note 1 SDNS /FRNS: steroid-dependent/frequently relapsing nephrotic syndrome, RTX: rituximab

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Fig. 3
figure 3

The transition of cellular immunity for each patient over time. A The B cell change in children with SDNS/FRNS treated with RTX, B The NK cell change in children with SDNS/FRNS treated with RTX, C The CD3 T-cell change in children with SDNS/FRNS treated with RTX, D The CD4 T-cell change in children with SDNS/FRNS treated with RTX, E The CD8 T-cell change in children with SDNS/FRNS treated with RTX, F The CD4 T-cell/CD8 T-cell change in children with SDNS/FRNS treated with RTX. Note 1 SDNS/FRNS: steroid-dependent/frequently relapsing nephrotic syndrome, RTX: Rituximab

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The levels of IgG, IgA, C4, and C3 in the enrolled children showed no significant differences before treatment, after the first dose, after the second dose(P > 0.05) (Fig. 4). The level of IgM significantly decreased after the second dose compared to before treatment and after the first dose(P < 0.05) (Fig. 2). Among 7 children, RTX-4, RTX-5, and RTX-7 showed a more pronounced decrease in IgM levels (Fig. 2), and these cases also had a higher number of infections.

Fig. 4
figure 4

The transition of humoral immunity for each patient over time. G The B cell change in children with SDNS/FRNS treated with RTX, H The IgM change in children with SDNS/FRNS treated with RTX, I The IgG change in children with SDNS/FRNS treated with RTX, J The IgA change in children with SDNS/FRNS treated with RTX, K The C4 change in children with SDNS/FRNS treated with RTX, L The C3 change in children with SDNS/FRNS treated with RTX. Note 1 SDNS/FRNS: steroid-dependent/frequently relapsing nephrotic syndrome, RTX: Rituximab

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Discussion

Clinical characteristics and diagnostic

The subjects of our study were primarily children with initial steroid sensitivity in INS who later developed dependency or late-onset resistance, and all experienced frequent relapses. Among them, males were in the majority, and the age group was predominantly children with normal physical development levels. Most of the duration of INS were relatively short, with most under 3 years. Additionally, one child with a 4-year disease course had a genetic test showing a point mutation at one locus with no clinical significance. Another child, with a 6-year disease course, had a renal pathophysiology of minimal change disease (MCD), and genetic tests revealed point mutations at two loci with no clinical significance. A third child, with a 8-year disease course, had FSGS but did not undergo genetic testing. Currently, most studies focus on children with steroid-dependent conditions [14, 15], while those with steroid resistance are more likely to relapse after RTX treatment [16], and this study also focused on steroid-dependent children. Furthermore, children with earlier onset ages are more likely to relapse after RTX treatment [17, 18], and as age increases, the number of relapses tends to decreases, but the effect may be more severe. Some children in this study had an earlier onset age, but RTX still achieved good efficacy, further follow-up observation is needed. Lastly, we have not yet identified potential histological factors as significant predictors of RTX treatment response to RTX treatment [16], and the impact of genetic changes is even less understood.

RTX efficacy and side effects

After receiving two doses of RTX, the study subjects took MMF orally to maintain remission once their B cell counts had recovered (≥ 5/ul), and none of them experienced a relapse within 12 months. All children in the study were able to taper off steroids within 6 months. Mason AE et al. suggested that children with secondary steroid resistance may respond well to RTX [19]. In this study, the children initially had SSNS, one of whom developed late resistance, but both achieved good outcomes. A randomized controlled clinical study by Basu B et al. demonstrated that RTX is more effective in maintaining remission and reducing steroid dosage in SDNS, and can be considered as a first-line treatment supplement to steroids [20]. Kari JA et al. showed that the efficacy of RTX in treating SDNS/FRNS is not inferior to cyclophosphamide, and further clinical studies are warranted to determine if it can be used as a first-line treatment [21]. An international multicenter study by Chan E et al. confirmed that repeated use of RTX in children with FRNS/SDNS can improve clinical outcomes, and while the side effects are acceptable, vigilance is required for serious adverse reactions. The study suggests that RTX is intended to be used as a first-line treatment [15]. This study utilized RTX in children with FRNS/SDNS, and observed good efficacy within 12 months. It is also hypothesized that RTX can be recommended as a first-line clinical treatment after a comprehensive assessment of the individual child’s situation. Girişgen İ et al. suggested that there is no necessity for urinary protein to turn negative before administering RTX, and considering that some RTX medication is lost from the urine during its use, it has been proposed that four doses can be considered to increase the effective amount of the drug for optimal efficacy [22]. Our study administered two doses of RTX after the children’s urinary protein turned negative and observed good efficacy within 12 months, suggesting that it is best to use RTX once the urinary protein is negative. Chan E Y-h et al. also emphasized that the dosage of RTX and the maintenance treatment with immunosuppressants are crucial for long-term maintenance of NS remission [23]. A randomized controlled clinical study by Ravani P et al. also confirmed the effect of RTX in maintaining remission of SDNS for an extended period, but found that low-dose MMF (350mg/m2) maintenance was not beneficial [5]. Iijima K et al. pointed out that MMF can fully prevent treatment failure after RTX treatment and has good tolerance, although its role in preventing relapses disappears after MMF is discontinued [10]. Thus, we assumed that two doses of RTX (375 mg/m2 per dose, once a week) with the standard oral dose of MMF (1000–1200 mg/m2 per day, divided into two doses) once B-cells recovered (≥ 5/ul) are effective in maintaining remission for children with SDNS/FRNS.

Additionally, our study subjects did not experience allergic reactions during the use of RTX. Enrolled children all took sulfamethoxazole tablets orally to prevent Pneumocystis pneumonia after use of RTX. During the 12-month follow-up period, aside from upper respiratory tract infections, the children did not exhibit any other serious infection symptoms. Among these cases, there were 4 instances of coronavirus infection, and the causative agents of the other infections remained unidentified, potentially linked to cross-infection exposure during the peak of COVID-19 pandemic. A randomized controlled clinical study by Chang D et al. also pointed out that the decrease in recurrence rate and reduction of steroid dosage in children with SDNS/SRNS were due to the use of RTX, but it did not increase the occurrence of adverse events [24]. An international multicenter study by Chan E et al. pointed out that repeated use of RTX in children with SDNS/FRNS can improve clinical outcomes, the side effects are acceptable, but vigilance is needed for serious adverse reactions [14]. This study found that some children were more susceptible to infect with B-cell depletion, particularly in conjunction with a decreasing trend in IgM levels-a phenomenon observed in cases 4, 5, and 7-yet without a corresponding decline in IgG levels. The rate of IgM level decreasing trend may correlate with B-cell depletion, but there may be other mechanisms at play that warrant further investigation. Thus, considering the finding form our study, the use of RTX to treat children with FRNS/SDNS is associated with minor side effects. However, caution must be exercised to prevent cross-infection of infectious diseases.

Dynamic changes in cellular and humoral immunity after RTX treatment

Children with FRNS/SDNS had B cell depletion after two doses of RTX within one week, and their B cells had recovered after 5–12 months. NK cell counts increased during the B cell depletion phase. However, as B cells regenerated, NK cells also increased gradually, while other cellular components of the immune system did not exhibit significant dynamic changes. Colucci M et al. pointed out that RTX can lead to complete depletion of B cells, which typically recover after 12 months without a corresponding change in T cell levels. And, the memory B cell subset significantly reduced, suggesting that monitoring memory B cells may potentially help predict relapses in NS [25]. Another study by Colucci M et al. confirmed that an increase in number of memory B cells relative to other B cell subsets may correlate with the onset of SSNS in children [26]. Ling C et al. suggested that a decrease in the ratio of transitional B/memory B is associated with the relapse of SSNS, and this ratio may serve as a useful predictor for relapses in children with SSNS [27]. Yang X et al. demonstrated that CD19+ CD138+ plasma cells may be a pivotal factor in the pathogenesis of pediatric nephrotic syndrome (PNS), whereas CD27+ memory cells might play a more significant role in the relapse of this disease [28]. Kim AHJ et al. indicated that B cells may mediate the production of urinary protein in children with nephrotic syndrome (NS) through the cytokine IL-4, thereby participating in the onset and progression of NS [29]. This study also indicates that RTX can deplete B cells with a relatively short recovery period, Unfortunately, the specific classification of the B cells was not provided, and suggests that NK cells may be implicated in the depletion of B cells, although the exact mechanism remains unclear. Boumediene A et al. showed that MCD in glomeruli involves a disruption of both innate and adaptive immune responses, which can be ameliorated by RTX treatment. However, this treatment results in no change in the expression of invariant killer NK cells (INKT) and a significant reduction in the number of Va24 chain (DN-TCR Va24) cells [30]. According to our study, an increase in NK cells coincides with the clearance of B cells. This is corroborated by the study of Kim AHJ et al., which supports the involvement of NK cells in the clearance of B cells. In contrast, the study by Boumediene A et al. did not observe any changes in NK cells, a discrepancy that may be attributed to the difference immune status in our study populations: ours consisted of subjects with SDNS/FRNS, while theirs had MCD pathology, or it could be related to the timing of the studies. We speculate that NK cells might, in the initial phase of B cell clearance, modulate cytotoxic effects through unknown cytokines, thereby participating in the clearance and subsequent recovery of B cells. The mechanism by which NK cells may be involved in the depletion of B cells is worth exploring.

Chen J et al. posit that both T cell and B cell dysfunctions are pivotal in the etiology of NS in children, akin to two sides of the same coin, yet the role of B cells appears to be more significant than that of T cells [13]. This study indicates that the counts of CD3 T cells, CD4 T cells, CD8 T cells, and the CD4 T/CD8 T cell ratio remained largely unchanged within 12 months post-RTX treatment. Chan CY et al. observed that after RTX treatment, there was a significantly reduction in the subsets of CD154+CD4+CD3+, IL-2+CD3+, and IFN-γ+CD3+ in children with FSGS, which are indicative of positive to RTX treatment and serve as promising predictive markers [31]. Zhang L et al. propose that γδT cells, IL-17γδT cells, and IL-23R+γδT cells may participate in the pathogenesis of PNS by modulating the Th17/Treg cell balance [32]. Another study by Colucci M et al. pointed out that the presence of atypical IgM on T cells can predict relapse and steroid dependence in NS [33]. CHAN CY et al. pointed out that early relapse following RTX treatment correlates with reduced baseline Treg levels and T cell hyporesponsiveness, suggesting that chronic T cell activation may be a valuable predictive marker. Forthermore, sustained NS remission is associated with the downregulation of Th2 cytokines [34]. Thus, the role of T cell function post-RTX treatment in children with NS warrants further clarification.

Colucci M et al. indicated that in children with FRNS/SDNS, both younger age and elevated levels of peripheral blood memory B cells are significant risk factors for relapse following RTX treatment, and the early recovery of memory B cells can serve as an independent predictor for relapse [35]. Al-Aubodah T-A et al. demonstrated that extrafollicular B cell responses are a defining characteristic of childhood INS, offering evidence for the extrafollicular origin of humoral immunity in active INS [36]. This study suggests that IgM levels gradually decrease in children with FRNS/SDNS following RTX treatment, whereas other parameters such as IgG, IgA, C3, and C4 do not exhibit significant dynamic alterations. Trachtman H et al. pointed out that IgM can activate the classical complement system within the glomerular of children with NS, potentially leading to glomerular damage [37]. Dossier C et al. indicated that treatment of refractory SDNS with obinutuzumab and daratumumab can prolong the depletion of peripheral B cells and remission, with some cases showing a reduction in peripheral blood IgM levels [38]. In summary, the early decrease in B cells following RTX treatment in children with FRNS/SDNS may suppress IgM production, which may be beneficial.

Limitations of the study

This study conducted a 12-month follow-up of the subjects, dynamically evaluating the efficacy and side effects in children, and monitored both cellular and humoral immune functions before and after RTX treatment. The novel findings indicate that administering two doses of RTX (each at a dosage of 375mg/m2, with one dose per week) followed by the addition of standard-dose MMF (1000-1200mg/m2/day, divided into two oral doses) has positive effects on SDNS/FRNS with minimal side effects. Furthermore, the proliferation of NK cells may contribute to the depletion of B cells, and the early depletion of B cells may potentially suppress the production of IgM. However, the numbers are relatively small, and no additional follow-up of humoral immune markers was performed two weeks after the second dose of RTX. The B cells and NK cells monitored were not further classified in detail, and additional clinical cases along with more in-depth monitoring of immune markers are necessary to clarify the immunological mechanisms underlying RTX treatment for INS. We are currently preparing to incorporate more cases and detailed immunological markers for further analysis.

Conclusion

Two doses of RTX (375mg/m2 per dose, 1 dose per week), along with standard dose of MMF (1000–1200/m2/d, divided into 2 doses orally) have proven effective in maintaining remission for children with SDNS/FRNS, with minimal side effects. B cells can be depleted one week after the second dose of RTX, and typically recover 5–12 months after the first of dose of RTX treatment. NK cell proliferation may play a role in B cell depletion, and early B cell depletion may suppress IgM production. These findings require further validation through additional clinical trials and basic research.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

SSNS:

Steroid-Sensitive Nephrotic Syndrome

RTX:

Rituximab

SDNS:

Steroid-Dependent Nephrotic Syndrome

FRNS:

Frequently Relapsing Nephrotic Syndrome

MMF:

Mycophenolate Mofetil

INS:

Idiopathic Nephrotic Syndrome

SRNS:

Steroid-Resistant Nephrotic Syndrome

FSGS:

Focal Segmental Glomerulosclerosis

MCD:

Minimal Change Disease

SMZ + TMP:

Sulfamethoxazole + Trimethoprim

PCR:

Protein-to-Creatinine Ratio

eGFR:

Estimated Glomerular Filtration Rate

PNS:

Pediatric Nephrotic Syndrome

NS:

Nephrotic Syndrome

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Acknowledgements

We thank all the participants who took part in our study, and pharmacists, nurses, laboratory technicians, statisticians, volunteers, receptionists of our study and our family for their supports and dedications.

Funding

This study was supported by grants 2021YFC2500202 from National Key Research and Development Program of China.

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Author notes

  1. Songlei Gu and Tong Shen are joint first authors.

Authors and Affiliations

  1. Department of Pediatrics, Women and Children’s Hospital, School of Medicine, Xiamen University, Zhenhai Road 10, Xiamen, Fujian, 361102, China

    Songlei Gu, Tong Shen, Jie Niu, Wenli Xu, Yugui Zeng & Xiaoqing Yang

  2. Department of Nephrology, Children’s Hospital of Fudan University, Wanyuan Road 399, Shanghai, 201102, China

    Yihui Zhai, Qian Shen & Hong Xu

  3. Pediatrics Department, Nanping Zhenghe County General Hospital, Shuinan Middle Road 69, Nanping, 353600, China

    Jie Yu

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  1. Songlei Gu
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Contributions

Songlei Gu, Tong Shen, Xiaoqing Yang, and Hong Xu conceptualized the study concept and design. Songlei Gu, Jie Yu, Jie Niu, Wenli Xu and Yugui Zeng conducted research. Songlei Gu analyzed the data. Songlei Gu, Yihui Zhai and Qian Shen drafted the manuscript. All authors were involved in writing the paper and had final approval of the submitted and published versions.

Corresponding authors

Correspondence to Hong Xu or Xiaoqing Yang.

Ethics declarations

Ethics approval and consent to participate

The study adhered to the Declaration of Helsinki. All procedures were approved by the medical ethics committee of Women and Children’s Hospital, School of Medicine, Xiamen University (application number: KY-2024-114-K01). The parents were informed of the project aims and requirements. All participates were below 16 years in the study. Informed consent was obtained from the parents of the participants (below 16 year).

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Not applicable.

Competing interests

The authors declare no competing interests.

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Gu, S., Shen, T., Zhai, Y. et al. The efficacy and dynamic changes of immune function of rituximab with mycophenolate mofetil in the treatment of steroid-dependent /frequently relapsing nephrotic syndrome: a retrospective follow-up study. BMC Nephrol 26, 186 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12882-025-04093-4

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Keywords

BMC Nephrology

ISSN: 1471-2369

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