Efficacy, Safety, And Mechasim Of Rituximab In Children With Frequently Relapsing/Steroid-Dependent Nephrotic Syndrome

Introduction Frequently relapsing(FR)/steroid-dependent(SD)nephrotic syndrome(NS)follows a relapsing and remitting course.It is also characterized by proteinuria and edema,which can significantly affect health-related quality of life(HRQOL)in children.Frequently relapsing/steroid-dependent nephrotic syndrome(FRSDNS)leads to steroid toxicity impairing the health-related quality of life(HRQOL),thus prompting the use of steroid-sparing drugs.This study evaluated the effectiveness and safety of a single dose of rituximab(RTX)as well as the impact of RTX on HRQOL in children with FRSDNS.This study compared the effectiveness and safety of rituximab,cyclophosphamide,and tacrolimus as first-line steroid-sparing agents and their impact on the HRQOL in children with FRSDNS.The podocytes were injured using puromycin aminonucleoside(PAN),then intervented with rituximab(RTX)and/or methylprednisolone(MP).The study aimed to observe the expression of transient receptor potential cation channel 6(TRPC6),apoptosis rate,and culture supernate levels of IL-1? and IL,18 in podocytes at 8h,24h,and 48h.Materials and Methods Sixteen children with FRSDNS were enrolled in the study.Each patient was administereed a single intravenous dose of RTX(375 mg/m2).Effectiveness was defined as remission of proteinuria.The side effects of RTX were monitored.Fifty-one children with FRSDNS not previously treated with steroid-sparing agents between 2019 and 2020 were randomized to receive rituximab(single dose of 375 mg/m2),standard cyclophosphamide,or tacrolimus,along with a tapering dose of prednisolone.Before and after treatment,clinical findings and side effects were evaluated.HRQOL was estimated using the PedsQLTM 4.0 Generic Core Scales.Mouse podocyte cell line(MPC5)were cultured in vitro.CCK-8 examined cell viability and the optimum concentrations of PAN,MP,and RTX.PAN induced podocyte injury.After MP and/or RTX intervention,the apoptosis rate,culture supemate levels of IL-1?and IL-18,mRNA and protein expression of TRPC6 in podocyte were detected by flow cytometry,enzyme-linked immunosorbent assay(ELISA),quantitative real-time PCR(qRT-PCR)and Western blotting,respectively.Results(1)All the patients completed the study.Three SDNS patients and three FRNS patients discontinued treatment over 1-3.25 years of follow-up.Additionally,three SDNS patients and three FRNS patients experienced 1-2 relapses.The mean relapse-free period was(79.00 ± 77.64)days.The mean dosages of prednisolone and other immunosuppressants required were significantly lower(P<0.05,<0.001)six months aftar treatment with RTX compared with six months before treatment.Relapse rate was significantly reduced(P<0.001)after treatment with RTX.Skin rash,hypotension,and fever were observed in one child.Total health score and physical,emotional,and school functioning were significantly higher six months after treatment with RTX(all P<0.001).(2)The mean relapse rate in all groups declined six months after treatment(P<0.05).The one-year relapse-free survival rate,number of relapses,and cumulative prednisolone dosage were lower with rituximab than with tacrolimus and cyclophosphamide(all P<0.05).The mean time to first relapse was 8.3,4.6,and 3.3 months with rituximab,tacrolimus,and cyclophosphamide,respectively.Although all treatments were well tolerated,the cyclophosphamide group had twice the frequency of infections compared to the other groups(P=0.001)At one year after treatment,the total,psychological health summary,and social and school functioning scores showed greater improvement in the rituximab group compared to the other groups(all P<0.05).(3)At 8h,24h,and 48h,CCK-8 found that PAN(50?g/mL)had obvious cell varibility,and it could be used as the optimum concentrations to induce podocyte injury;MP(100 ng/mL)and RTX(100 ?g/mL)showed little cell varibility,and they were the best concentration.qRT-PCR and Western blotting found that TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group(all P<0.05).Compared with PAN group,MP,RTX,and MP+RTX groups decreased TRPC6 protein expression at 24h and 48h and TRPC6 mRNA at 3 time-points(all P<0.05).In PAN group,the apoptosis rates were significantly increased at 24h and 48h(all P<0.05).MP and RTX significantly prevented the shrinkage and apoptosis of podocytes compared with PAN group at 24h and 48h(all P<0.05).At 3 time-points,the levels of IL-1? and IL-18 in PAN group were significantly higher than those in control group(all P<0.05),and PAN group had higher levels of IL-1? and IL-18 compared to MP group(all P<0.05).Levels of IL-1? and IL-18 were lower in RTX group than in PAN group at 24h and 48h(all P<0.05).Conclusion(1)A single dose of RTX is effective and safe for children with FRSDNS and can improve HRQOL,especially physical,emotional,and school functioning.(2)Rituximab is more effective and safer than cyclophosphamide and tacrolimus as a first-line steroid-sparing agent in children with FRSDNS.It improves the HRQOL,especially psychological,social,and school functioning.(3)MP and RTX could protect podocyte and prevent proteinuria by decreasing the expression of TRPC6 mRNA and protein,reducing podocyte apoptosis,inhibiting the expression of inflammatory.The First Part Single dose of rituximab in children with frequently relapsing/steroid-dependent nephrotic syndrome:Clnical efficacy and evaluation of health-related quality of lifeIntroduction Frequently relapsing(FR)/steroid-dependent(SD)nephrotic syndrome(NS)follows a relapsing and remitting course.It is also characterized by proteinuria and edema,which can significantly affect health-related quality of life(HRQOL)in children.This study evaluated the effectiveness and safety of a single dose of rituximab(RTX)as well as the impact of RTX on HRQOL in children with FRSDNS.Materials and Methods Sixteen children with FRSDNS were enrolled in the study.Each patient was administered a single intravenous dose of RTX(375 mg/m2).Effectiveness was defined as remission of proteinuria.The side effects of RTX were monitored.HRQOL was assessed using PedsQLTM 4.0 Generic Core Scales.Results(1)All the patients completed the study.Three SDNS patients and three FRNS patients discontinued treatment over 1-3.25 years of follow-up.Additionally,three SDNS patients and three FRNS patients experienced 1-2 relapses.The mean relapse-free period was(79.00±77.64)days.(2)The mean dosages of prednisolone and other immunosuppressants required were significantly lower(P<0.05,<0.001)six months after treatment with RTX compared with six months before treatment.Relapse rate was significantly reduced(P<0.001)after treatment with RTX.(3)Skin rash,hypotension,and fever were observed in one child.(4)Total health score and physical,emotional,and school functioning were significantly higher six months after treatment with RTX(all P<0.001).Conclusion A single dose of RTX is effective and safe for children with FRSDNS and can improve HRQOL,especially physical,emotional,and school functioning.The Second Part Comparison of rituximab,cyclophosphamide,and tacrolimns as first steroid-sparing agents for complicated relapsing/steroid-dependent nephrotic syndrome in children:An evaluation of the health-related quality of lifeBackground Frequently relapsing/steroid-dependent nephrotic syndrome(FRSDNS)leads to steroid toxicity impairing the health-related quality of life(HRQOL),thus prompting the use of steroid-sparing drugs.This study compared the effectiveness and safety of rituximab,cyclophosphamide,and tacrolimus as first-line steroid-sparing agents and their impact on the HRQOL in children with FRSDNS.Methods Fifty-one children with FRSDNS not previously treated with steroid-sparing agents between 2019 and 2020 were randomized to receive rituximab(single dose of 375 mg/m2),standard cyclophosphamide,or tacrolimus,along with a tapering dose of prednisolone.Before and after treatment,clinical findings and side effects were evaluated.HRQOL was estimated using the PedsQLTM 4.0 Generic Core Scales.Results(1)The mean relapse rate in all groups declined six months after treatment(P<0.05).(2)The one-year relapse-free survival rate,number of relapses,and cumulative prednisolone dosage were lower with rituximab than with tacrolimus and cyclophosphamide(all P<0.05).The mean time to first relapse was 8.3,4.6,and 3.3 months with rituximab,tacrolimus,and cyclophosphamide,respectively.(3)Although all treatments were well tolerated,the cyclophosphamide group had twice the frequency of infections compared to the other groups(P=0.001).(4)At one year after treatment,the total,psychological health summary,and social and school functioning scores showed greater improvement in the rituximab group compared to the other groups(all P<0.05).Conclusion(1)Rituximab is more effective and safer than cyclophosphamide and tacrolimus as a first-line steroid-sparing agent in children with FRSDNS.(2)It improves the HRQOL,especially psychological,social,and school functioning.The Third Part Ritnximab and methylprednisolone ameliorate PAN-induced podocyte injury through inhibiting the expression of transient receptor potential cation channel 6Objective The podocytes were injured using puromycin aminonucleoside(PAN),then intervented with rituximab(RTX)and/or methylprednisolone(MP).The study aimed to observe the expression of transient receptor potential cation channel 6(TRPC6),apoptosis rate,and culture supernate levels of IL-1? and IL-18 in podocytes at 8h,24h,and 48h.Methods Mouse podocyte cell line(MPC5)were cultured in vitro.CCK-8 examined cell viability and the optimum concentrations of PAN,MP,and RTX.PAN induced podocyte injury.After MP and/or RTX intervention,the apoptosis rate,culture supernate levels of IL-1? and IL-18,mRNA and protein expression of TRPC6 in podocyte were detected by flow cytometry,enzyme-linked immunosorbent assay(ELISA),quantitative real-time PCR(qRT-PCR),and Western blotting,respectively.Results(1)At 8h,24h,and 48h,CCK-8 found that PAN(50?g/mL)had obvious cell varibility,and it could be used as the optimum concentrations to induce podocyte injury;MP(100 ng/mL)and RTX(100 ?g/mL)showed little cell varibility,and they were the best concentration.(2)qRT-PCR and Western blotting found that TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group(all P<0.05).Compared with PAN group,MP,RTX,MP+RTX groups decreased TRPC6 protein expression at 24h and 48h and TRPC6 mRNA at 3 time-points(all P<0.05).(3)In PAN group,the apoptosis rates were significantly increased at 24h and 48h(all P<0.05).MP and RTX significantly prevented the shrinkage and apoptosis of podocytes compared with PAN group at 24h and 48h(all P<0.05).(2)At 3 time-points,the levels of IL-1? and IL-18 in PAN group were significantly higher than those in control group(all P<0.05),and PAN group had higher levels of IL-1? and IL-18 compared to MP group(all P<0.05).Levels of IL-1? and IL-18 were lower in RTX group than in PAN group at 24h and 48h(all P<0.05).Conclusion MP and RTX could protect podocyte and prevent proteinuria by decreasing the expression of TRPC6 mRNA and protein,reducing podocyte apoptosis,inhibiting the expression of inflammatory factors.