Introduction

Hepatitis C virus (HCV) infection is a global health problem, with an estimated 71 million people chronically infected with HCV. In 2015, the global incidence of HCV was 23.7 cases per 100,000 population, and the estimated number of new diagnoses of HCV infection in 2015 was 17.5 million1. Despite the availability of effective anti-HCV drugs, there is a risk for reactivation of HCV in patients with resolved HCV infection with immunosuppressive therapy2,3.

It has been demonstrated that although rituximab greatly improved the prognosis of patients with hematological malignancies and rheumatoid arthritis, it is also associated with HCV reactivation4,5. Recently, rituximab has emerged as an effective treatment option for patients with phospholipase A2 receptor (PLA2R) associated membranous nephropathy (MN)6. However, despite the increasing use of rituximab, there is a lack of studies related to HCV reactivation in patients with PLA2R associated MN and resolved HCV infection receiving rituximab. With the increased use of rituximab in patients with PLA2R associated MN, there is an urgent need to determine the risk of HCV reactivation in these populations. Therefore, we included a group of patients with PLA2R associated MN in combination with resolved HCV infection treated with rituximab to determine the efficacy and safety of rituximab treatment in these patients, especially the risk of HCV reactivation.

Methods

Study design and patients

This is a retrospective study. A total of 598 adult patients with PLA2R associated MN who received rituximab at the First Affiliated Hospital of Zhengzhou University from January 2019 to December 2021 were screened. We identified 8 cases of PLA2R associated MN with resolved HCV infection. A diagnosis of resolved HCV infection was serum positivity with HCV RNA undetectable.

The exclusion criteria were as follows: (1) patients together with additional glomerular diseases including secondary membranous nephropathy; (2) patients who had poor follow-up compliance while receiving rituximab; (3) patients with a follow up period less than 12 months; (4) patients who were co-infection with hepatitis B virus; (5) patients who received other immunosuppressive agents except for rituximab simultaneously; (6) patients who are with cryoglobulinemia or hypocomplementemia. Informed consent was derived from the participants. Treatment regimens for all patients were based on the recommendations in relevant guidelines6. We also reviewed the guidelines about the study participants to confirm that our study was conducted in accordance with the relevant guidelines/regulations. The ethics committee of the First Affiliated Hospital of Zhengzhou University approved the study.

Immunosuppressive regimen and follow-up

The patients in this study all received a regimen of rituximab (375 mg/m2 × 4 doses or 1 g × 2 doses). RTX was redissolved in normal saline to a final concentration of 1 mg/mL, which was then infused at an initial rate of 20 mL/h, gradually increasing to 100 mL/h according to the tolerance of each patient. At 6 months, if the reduction in anti-PLA2R antibodies is between 50 and 90%, RTX therapy will continue.

General clinical information, including gender, age, , pathological information, and past treatment plans were gathered from medical records. Routine blood examinations, liver and kidney function tests, blood lipids, 24-h urine protein, anti-PLA2R antibody, circulating B-cell quantity, and HCV viral titers were collected at the time of rituximab infusion and repeated at 1–3 months interval after rituximab administration. According to age, gender, race, and serum creatinine levels, the estimated glomerular filtration rate (eGFR) was determined using the Chronic Kidney Disease Epidemiology Collaboration algorithm.

Definitions and monitoring of HCV reactivation

HCV reactivation was defined as the reappearance of HCV RNA after rituximab treatment. All patients were screened for HCV (HCVAb and HCV RNA) prior to rituximab treatment and monthly after receiving rituximab, along with routine liver function tests. Serum HCVAb was determined by electrochemiluminescence using the Cobase immunoassay analyzer and the immunoassay "ECLA" (Roche Diagnostic, Germany). Plasma HCV-RNA virus load was determined using real-time polymerase chain reaction on the ABI7500 Fluorescent Quantitative PCR Instrument (Applied Biosystems, USA). The lower limit of HCV RNA detection was 500 IU/mL in our study.

Treatment response

According to the 2021 Kidney Disease Improving Global Outcomes recommendations, complete remission was defined as urinary protein excretion < 0.3 g/24 h with normal serum albumin and serum creatinine. Partial remission was defined as proteinuria < 3.5 g/24 h or a reduction of ≥ 50% from peak, improvement or normalization of serum albumin, and stable serum creatinine. Immunologic remission was defined as the titer of anti-PLA2R antibody below 2 RU/ml.

Statistical analysis

Data was expressed as mean ± standard deviation, median, interquartile range, or percentages. Paired sample t-test or the Wilcoxon matched pair signed-rank (two samples) test was used for comparison between the two groups according to the distribution of data. The SPSS 24.0 software program was used for statistical analysis, and two-sided p-values were calculated.

Results

Baseline characteristics at rituximab infusion

There were 8 patients with PLA2R associated MN and resolved HCV infection who received rituximab treatment. The clinical characteristics of the patients at baseline was presented in Table 1. There were 5 male patients and 3 female patients, with a mean age of 50.13 ± 4.29 years old. Anti-PLA2R antibody was positive in all 8 patients. The baseline level of proteinuria was 5.29 (3.53, 6.51) g/24 h, serum albumin was 28.00 (25.05, 32.55) g/L, and eGFR was 71.10 (48.54, 89.97) ml/min/1.73 m2. According to the chronic kidney disease staging guidelines of Kidney Disease: Improving Global Outcomes, 2, 3, and 3 patients had stage 1, 2, and 3 chronic kidney disease, respectively.

Table 1 Baseline characteristics of MN patients with HCV infection at RTX infusion.
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Following diagnosis by percutaneous renal biopsy, all patients were administered other immunosuppressant therapy before rituximab treatment, including cyclophosphamide combined with steroids in 2 patients, cyclosporine combined with steroids in 1 patient, and tacrolimus in 8 patients. The mean time from initial first-line immunosuppressive therapy to initial RTX therapy was 7.88 ± 4.67 months. In previous immunosuppressive treatment regimens, 5 patients achieved complete remission. However, during drug discontinuation or reduction, nephrotic syndrome relapsed. The other 3 patients did not achieve partial or complete remission of nephrotic syndrome in previous immunosuppressive treatment regimens.

Histological findings

All patients underwent kidney biopsy and were diagnosed with PLA2R associated MN. The renal biopsy findings are detailed in Table 2. LM showed a median of 33 glomeruli per biopsy (range 15–73). Interstitial fibrosis and tubular atrophy were present in 3 cases. Immunostaining revealed granular glomerular capillary wall positive for IgG and C3 in all patients. PLA2R and IgG4 staining was tested by renal biopsy staining and positive in all 8 cases. EM was performed in all patients and showed features of MN with subepithelial electron dense deposits. There were mostly Stage I-II and II-III and the remaining cases showed deposits of stage II. Tubuloreticular inclusions were not found in all patients. Mesangial, intramembranous, and subendothelial deposits were not seen in all 8 patients.

Table 2 Pathological characteristics of patients.
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Basic information of HCV infection

As shown in Table 3, in 5 patients, the diagnosis of HCV infection predated the renal biopsy, and in 3 patients the diagnosis of HCV infection and PLA2R associated MN was made at the same time. All 8 patients with previous HCV infection received antiviral agents and the load of HCV virus were undetectable at the time of rituximab treatment. None of the 8 patients had cryoglobulinemia and hypocomplementemia. (Table 1).

Table 3 Clinical findings in MN patients with hepatitis C.
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Clinical and immunological outcomes

During the follow-up period of 19.00 (16.00, 25.25) months, the prevalence of patients with clinical remission increased from 0.00% at baseline to 50.00% (4/8), 75.00% (6/8), and 87.50%(7/8) at month 6, month 12, and last visit. The median CD19 + B cell count was 292/mm3 (IQR, 188.00 to 327.75) at baseline. CD19 + B cell counts at month 6 were significantly reduced compared with the baseline level (P = 0.017). At month 12 and the last visit, the number of CD19 + B cells had recovered to some extent. However, significant differences in CD19 + B cell counts remained between the baseline level and the levels at month 12 (P = 0.028) and the last visit (P = 0.018), respectively, after rituximab intervention (Table 4, Fig. 1).

Table 4 Efficacy outcome variables.
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Fig. 1
figure 1

Serial levels of proteinuria (A), serum albumin (B), serum creatinine (C), eGFR (D), CD19 positive B cells (E), anti-PLA2R antibody (F), ALT (G), AST (H), and TBIL (I) after the rituximab treatment in all patients who had been followed up for a minimum of 12 months. The bars show median with interquartile range for each variable. ns P > 0.05 vs baseline; *P < 0.05 vs baseline; **P < 0.01 vs baseline; ***P < 0.001 vs baseline.

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At month 6, month 12, and last visit, anti-PLA2R antibody titers [6.60 (2.00, 18.00) vs. 34.20 (18.20, 85.30) RU/ml, P = 0.059; 2.00 (2.00, 2.50) vs. 34.20 (18.20, 85.30) RU/ml, P = 0.028; 2.00 (2.00, 2.00) vs. 34.20 (18.20, 85.30 RU/ml, P = 0.018] were lower compared with the baseline levels (Table 4, Fig. 1). From baseline to month 6, month 12, and last visit, there was a decrease in prevalence of anti-PLA2R positive patients from 100.00% (8/8) to 50.00% (4/8), 12.50% (1/8), and 0.00% (0/0).

In term of proteinuria, the results showed that median levels of proteinuria were decrease from 5.29 (3.53, 6.51) g/d at baseline to 1.10 (0.38, 1.85) g/d at month 12 and 0.62 (0.19, 0.91) g/d at last visit, respectively (P = 0.041, P = 0.028). At month 6, serum albumin increased significantly [27.30 (20.20, 34.40) vs 23.70 (18.70, 25.70) g/L, P = 0.017] compared with the baseline levels. Continued improvements of serum albumin were observed between baseline and last visit (Table 4, Fig. 1).

Renal function was preserved since rituximab infusion in all patients demonstrated by serum creatinine and eGFR. This phenomenon also showed that the reduction of proteinuria caused by rituximab might maintain the stability of renal function to some extent (Table 4, Fig. 1).

Safety analysis of rituximab in patients with PLA2R associated MN and HCV infection

This study showed the safety of rituximab in patients with PLA2R associated MN and resolved HCV infection, which was demonstrated by no increase in HCV virus load and stable liver function tests after rituximab therapy (Fig. 1). During the follow-up, we also did not observe other adverse events including the occurrence of allergic reaction and infection.

Discussion

In this study, we retrospectively analyzed demographic characteristics, clinical remission rate, liver function and HCV load changes in patients with PLA2R associated MN and resolved HCV infection treated with rituximab.

Immunosuppression is known to increase HCV virus load and accelerate the progression of chronic HCV liver disease7,8,9, and previous studies suggested a similar risk with rituximab treatment10. This limited the application of rituximab in patients with PLA2R associated MN and resolved HCV infection. However, unlike HBV and HIV, HCV infection can be completely and permanently cured through antiviral therapy, as HCV does not have a long-term storage in the body. Furthermore, the widespread and effective application of antiviral drugs in clinical practice brings new hope to the application of rituximab in patients with PLA2R associated MN and resolved HCV infection in recent years.

At present, there are no studies about the safety of rituximab in patients with PLA2R associated MN and resolved HCV infection. However, in other diseases such as lymphoma and other HCV-associated nephritis, the results of the studies are inconsistent as to whether rituximab affects the replication of HCV. To date, in very small cohorts of patients with haematological malignancies receiving R-CHOP regimens, results had showed the elevated serum HCV virus load during or after rituximab-based chemotherapy11,12,13. In a larger cohort study of patients with HCV-associated diffuse large B-cell lymphoma14, serum HCV virus load increased significantly during rituximab-based chemotherapy and decreased significantly thereafter. There were also some studies similar to ours that confirm the safety of rituximab. A prospective controlled trial showed that among 31 patients with severe HCV associated cryoglobulinemia vasculitis who were randomly treated with rituximab and antiviral therapy, the complete remission rate of kidney disease was high and the safety was good15. Consistent with the above studies, other reports have shown the safety of rituximab in HCV infected individuals. These reports showed that there was no increase in HCV viremia after rituximab treatment, and liver function tests were stable16.

In our trial, we found no evidence of HCV reactivation . There were no significant changes in plasma viral levels over time and no biochemical evidence of hepatitis flare. Rituximab treatment was well tolerated, and no serious infections were detected. We considered that it may be related to the following reasons. Firstly, in the previous studies mentioned above, most of patients still had a high HCV virus load at rituximab infusion, and these patients also received cyclophosphamide, doxorubicin, vincristine, and prednisolone treatment when receiving rituximab treatment, which may be the reason for the increase of HCV virus load. In our study, patients received antiviral therapy before receiving rituximab treatment, and HCV virus was not detected at rituximab infusion. Moreover, in our study, patients did not receive other immunosuppressants except for rituximab, which may be one of the reasons why our results differ from some previous studies.

Our current study has several limitations. First, because the study was a retrospective study and the number of cases included in the study was low, our results should be interpreted with caution and should be further evaluated in a large study of prospective design. Second, due to the small sample size, we did not compare the difference in HCV viral load between patients treated with rituximab and those not treated with rituximab. Whether rituximab is an independent factor contributing to the increase of HCV viral load in patients with PLA2R associated MN is unknown and should be investigated in further studies.

In conclusion, our findings suggest that rituximab does not lead to a significant increase in HCV virus load in patients with PLA2R associated MN and resolved HCV infection. On the basis of successful eradication of HCV virus with antiviral drugs, rituximab may be an effective regimen for treating patients with PLA2R associated MN with resolved HCV infection. However, this finding is based on a very small sample size and should be confirmed in larger clinical trials.