CKD: Chronic Kidney Disease; CSA: Cylosporine A; IA: Immunosuppressive Agents; MMF: Mycofenolate Mofetil; RTX: Rituximab; SRNS: Steroid Resistant Nephrotic Syndrome

Steroid Resistant Nephrotic Syndrome (SRNS) is a rare but serious condition defined as the lack of complete remission at 4 weeks daily prednisolone therapy [1]. Its prevalence varies depending on the population and geographical region studied. However, it is estimated that 10-20% of children with nephrotic syndrome will be steroid-resistant [2]. The management of SRNS often requires the use of immunosuppressive agents (IA) including Cyclosporine A (CsA), Mycophenolate Mofetil (MMF), and rituximab (RTX) [3]. CsA is a calcineurin inhibitor that inhibits T-cell signaling in lymphocytes and also has direct non-immune effects on the podocyte actin cytoskeleton. MMF inhibits inosine monophosphate dehydrogenase, a key enzyme in purine biosynthesis, suppressing DNA replication in T and B lymphocytes to modulate the immune response. RTX is a chimeric monoclonal antibody that targets CD20 on B lymphocytes, depleting them to regulate the immune system. It also stabilizes SMPDL-3b, preventing podocyte actin remodeling, although this is not related to the immune system [2,3,12]. While these IA have shown efficacy in the treatment of SRNS, there is still much controversy about the optimal use and combination of these drugs. In particular, the use of therapy with two or more simultaneous IA, has been suggested as a potential strategy to improve the outcomes of patients with SRNS. The aim of this study is to describe the characteristics and disease course of SRNS in children who have received combination therapy with CsA, MMF, and RTX. This study seeks to provide insights into the use of IA in the management of this challenging condition.

The study design was that of a retrospective single-center chart including patients < 18 years with SRNS, who were treated at a pediatric tertiary care department between January 2008 and December 2020. Subjects with congenital or infantile or secondary nephrotic syndrome were excluded as well as those with systemic disease, vasculitis or glomerulonephritis. Were included those who were treated with an association of at least two IA including CsA, MMF, or RTX, with a minimal required follow-up period of one year. Data were collected for IA initiation time, duration of use, response rate and toxicity for each treatment. Drug response was recorded for those with 2 or more months of immunosuppressant treatment.

Definitions: The cases were defined according to Kidney Disease Improving Global Outcome (KDIGO) nomenclature of glomerular disease [1]:

- Steroid resistance was defined as lack of response to a course of corticosteroid therapy: prednisone or prednisolone at a dose of 60 mg/m² of body surface area per day for 4 weeks.

- Complete remission is defined as a urinary protein excretion of ≤ 0.3 g/24 hours, with normal serum albumin levels and resolution of edema.

- Partial remission is defined as a urinary protein excretion of ≤ 3.5 g/24 hours, with a reduction of at least 50% from baseline proteinuria, and stable or improved serum albumin levels.

- Relapse is defined as a return of protein excretion ≥ 50mg/kg/24 hours or more in a patient who previously achieved complete or partial remission for at least three consecutive days after reduction or discontinuation of immunosuppressive therapy.

- CsA resistance was defined as lack of response to CsA after 6 months of treatment

The following clinical data were recorded: gender, family history of nephrotic syndrome, age of disease onset, clinical and biological characteristics at the diagnosis of nephrotic syndrome, renal function, histopathological type and genetic findings, age of CsA initiation, relapses while on CsA and adverse effects, time to initiation of the second and/or third IA and adverse effects, and disease course using two or more IA. All patients were treated according to the protocol of the French Society of Pediatric Nephrology [4]. Children presenting with SRNS were initially treated with Cs A at a dose of 150-200 mg/m² of body surface area per day, with a target residual level between 100 and 300 ng/mL, along with prednisone at a dose of 30 mg/m² of body surface area per day for 1 month, followed by 30 mg/m² of body surface area on alternate days for five months.

The response to therapy was evaluated at the 4-month mark following the initiation of treatment. If complete or partial remission had not been achieved, the treatment was discontinued. In cases where remission was obtained, the dosage of prednisone was gradually decreased and discontinued within the next 3 months, while that of CsA was tapered and terminated in the following 3 months (with a total treatment duration of 12 months). The dose of CsA was adjusted according to the residual rate and any adverse effects. After treating or excluding any chronic infection and verifying treatment observance and genetics result, nephrotic syndrome was defined as refractory. So, second-line agents were introduced MMF and/or RTX. MMF was used at the dose of 600mg/m² of body surface/day in two divided doses for 12-36 months [1]. RTX was used at the dose of 375mg/m² of body surface/ injection twice [1].

Statistical analysis

Qualitative data were compared Pearson chi-square test. Means of quantitative data were compared using t-test. Cumulative rates were compared using Kaplan Meier survival function with log rank. A P value of < 0.05 was considered as significant. The primary outcome variable was the number of patients who went into remission (complete or partial). The secondary outcome variables were mean time to remission (after treatment initiation), toxicity rates relative to each immunosuppressive regimen and renal function (estimated glomerular filtration calculated with the modified Schwartz equation).

A total of 26 patients were included in the study, all of whom were on at least two IA. Corticosteroid resistance was noted at diagnosis in 36.4% of patients, while 63.6% became resistant later on. Demographic and clinical data of the cohort are shown in Table I. Renal biopsy was performed in 72% of patients. The most common histological finding was minimal change disease, observed in 36.4% of cases, followed by focal segmental glomerulosclerosis in 31.8% and diffuse mesangial proliferation in 4.5%. Genetic testing was performed in 40.9% of cases, with only one patient found to have the NPHS2 mutation.

The mean time of CsA initiation was 205 days: 67 days for those who presented with initially SRNS versus 287 days for those who were secondary SRNS. The mean initial CsA dosage was 160.80 ± 22.01 mg/day, and the mean residual level was 221.63 ± 109.93 ng/mL. The mean number of relapses under CsA was 2.53 ± 0.99. The time to initiation of a second IA was 48 months after the diagnosis of nephrotic syndrome. All patients in this study were treated with the combination of CsA and MMF, and two patients received the combination of CsA, MMF and RTX. The difference between mean delays to remission, after initiating CsA alone and under combined treatment with CsA and MMF or RTX; were respectively 6.71 ± 3 versus 4 ± 1.41 months (t-statistic=3.88; 95%CI: 1.3-4.11; P<10-3). Thus, cumulative remission rates who were detailed in survival curves in Figure 1, showed no significant difference between CsA and combined treatment (P=0.08).

Patients with SRNS who were treated by combined IA were compared a group of subjects under CsA (n=51): regrouping children on CsA alone before combining IA (n=26) and those who received exclusively CsA. The two groups were comparable. Different profiles of response to CsA and to combined IA were detailed in Table 2. The two patients treated with the combination of CsA, MMF and RTX achieved complete remission.

Treatment with IA was associated with toxicity in 65.5% of cases. CsA side effects were hypertrichosis in 63.6% of cases, gingival hypertrophy in 36.4% of cases, hypertension 40.9% of cases, and CKD in 31.8% of cases, side effects of MMF were mainly gastrointestinal disorders, noted in 43%. The patient who received the combination of CsA, MMF and RTX presented moderate lymphopenia. Thirteen patients developed severe renal disease which means stage 3, 4 or 5, among whom 23% (n=3) went to end-stage CKD. The mean time to progression to severe CKD was 45 ± 36 months after diagnosis. Overall, the mean creatinine at the end of the follow-up was up to 65.35 ± 23.17 µmol/L, which gave a mean estimated glomerular filtration rate of 76.40 ±37.38 ml/min/1.73m². This was significantly lower compared to the initial rates (t-statistic =6.69; 95% CI: 39.20-72.79; P <10-3) Figure 2.

The management of SRNS remains a clinical challenge due to the lack of consensus regarding optimal treatment strategies. Our study aimed to evaluate the efficacy and safety of a combination of two or more IA in the treatment of SRNS. Our results showed that all patients included in the study were on at least two immunosuppressive agents. The most commonly used IA was CsA, with MMF being the second-line most commonly used treatment. In a similar Colombian study testing multiple immunosuppressant therapies in SRNS, MMF was the most used (100%), followed by CsA (69.2%), Cyclophosphamide 23.1%, and RTX 23% [6].

The study demonstrated that a combination of CsA + MMF can lead to significant remission rates in patients with SRNS. In our cohort, 34.6% of patients achieved complete remission, which is consistent with the results of previous studies: Notably, patients who received the combination of CsA with both MMF and RTX achieved complete remission, suggesting that this combination may be a promising option for refractory cases of SRNS [3, 4]. In a recent review, Kamei and al reported that remission rates in patients with initial steroid resistance were 43.9% and late steroid resistance were 57.7%, while combining other secondline IA to RTX [7]. However, our study also revealed that the use of IA was associated with a significant rate of toxicity, particularly with the use of CsA alone or combined to MMF. Similar results were described in an Egyptian review, where these rates reached respectively 70.1%, 32.5 and 10.3%. [9] Others noted less frequent side effects with CsA: hypertrichosis in only 18 to 20% of cases and gingival hypertrichosis in only 7 to 15% [10].

Renal failure due to the use of CsA have been well described in literature. Ji Hong and al concluded that the median CsA residual level was an independent and significant risk factor for the development of CsA associated nephrotoxicity in children with nephrotic syndrome [8]. The time to progression to renal disease in this review underscores the importance of early detection and treatment of SRNS to prevent or delay CKD progression. Moreover, regular monitoring of renal function and blood pressure is crucial for patients with SRNS to detect and manage potential complications early on.

Despite the efficacy of the combination of CsA and MMF, a significant proportion of patients (15.38%) remained resistant to treatment. The reasons for treatment resistance are multifactorial and may include genetic factors, disease severity, and non-adherence to medication. In our cohort, genetic testing was performed in only 40.9% of cases, highlighting the need for broader genetic testing in patients with SRNS. Research has demonstrated that monogenic SRNS cases are associated with an increased resistance to immunosuppressive therapy. Therefore, genetic testing helps avoid these drugs and prevent potential adverse effects. This results in an added advantage of personalized treatment and the avoidance of renal biopsy [11].

Our study provides further evidence that a combination of CsA and MMF can lead to significant remission rates in patients with SRNS, although it is associated with a high rate of adverse effects. Future studies are needed to explore the efficacy and safety of newer IA and combination therapies in the treatment of SRNS. Recent studies has shown that the detection of individual genetic variations in SRNS has highlighted the importance of podocyte damage in causing the disease. Focusing on treating podocyte damage is crucial, and drugs that specifically target podocytes, along with immunosuppressants, are preferable as the primary treatment for SRNS [12].

SRNS is a rare and challenging condition that affects a subset of children with nephrotic syndrome. The management of SRNS requires the use of one or more IA, including CsA, MMF, and RTX. While these agents have shown efficacy, there is still controversy regarding the optimal use and combination these drugs. We described the disease course of children who received combination therapy with multiple associated IA. Our findings suggest that this combination therapy may be a viable treatment option. However, further studies are needed to determine the most effective and safe combination therapy for this challenging condition. The results of our study may provide insights into the use of IA for the management of SRNS and may guide the development of future treatment strategies.

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