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Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout.

ABSTRACT: Recently, recessive mutations of MAGI2 were identified as a cause of steroid-resistant nephrotic syndrome (SRNS) in humans and mice. To further delineate the pathogenesis of MAGI2 loss of function, we generated stable knockout lines for the two zebrafish orthologues magi2a and magi2b by CRISPR/Cas9. We also developed a novel assay for the direct detection of proteinuria in zebrafish independent of transgenic background. Whereas knockout of magi2b did not yield a nephrotic syndrome phenotype, magi2a-/- larvae developed ascites, periorbital edema, and proteinuria, as indicated by increased excretion of low molecular weight protein. Electron microscopy demonstrated extensive podocyte foot process effacement. As in human SRNS, we observed genotype/phenotype correlation, with edema onset occurring earlier in zebrafish with truncating alleles (5-6 days post fertilization) versus hypomorphic alleles (19-20 days post fertilization). Paradoxically, corticosteroid treatment exacerbated the phenotype, with earlier onset of edema. In contrast, treatment with cyclosporine A or tacrolimus had no significant effect. Although RhoA signaling has been implicated as a downstream mediator of MAGI2 activity, targeting of the RhoA pathway did not modify the nephrotic syndrome phenotype. In the first CRISPR/Cas9 zebrafish knockout model of SRNS, we found that corticosteroids may have a paradoxical effect in the setting of specific genetic mutations.

SUBMITTER: Jobst-Schwan T

PROVIDER: S-EPMC6481319 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout.

Jobst-Schwan Tilman T Hoogstraten Charlotte A CA Kolvenbach Caroline M CM Schmidt Johanna Magdalena JM Kolb Amy A Eddy Kaitlyn K Schneider Ronen R Ashraf Shazia S Widmeier Eugen E Majmundar Amar J AJ Hildebrandt Friedhelm F

Kidney international 20190305 5

Recently, recessive mutations of MAGI2 were identified as a cause of steroid-resistant nephrotic syndrome (SRNS) in humans and mice. To further delineate the pathogenesis of MAGI2 loss of function, we generated stable knockout lines for the two zebrafish orthologues magi2a and magi2b by CRISPR/Cas9. We also developed a novel assay for the direct detection of proteinuria in zebrafish independent of transgenic background. Whereas knockout of magi2b did not yield a nephrotic syndrome phenotype, mag ...[more]

PMID: 31010479

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Project description:Introduction:In last few years, several studies have revealed the remarkable stability of extracellular microRNAs (miRNAs) circulating in the blood or excreted in the urine and underscored their key importance as biomarkers of certain diseases. Since miRNA in urinary sediment is relatively stable and easily quantified, it has the potential to be developed as a biomarker for disease diagnosis and monitoring. Identification of serum and urinary levels of certain miRNAs may assist in the diagnosis and assessment of disease activity in patients with nephrotic syndrome (NS). The global expression profile of miRNAs in childhood NS in Indian population remains unknown. Hence, further research is warranted in this area. This study seeks to prospectively evaluate whether a multipronged multiomics approach concentrating on microRNA expression profiles in children with NS vis-a-vis normal healthy children is discriminant enough to predict steroid responsiveness in childhood NS. Methods and analysis:In this prospective multicentric cohort study, subjects will be recruited from general paediatric and paediatric nephrology outpatient departments (OPDs) in tertiary care level referral hospitals. Age-matched and sex-matched healthy individuals with normal renal function (as assessed by normal serum creatinine and normal ultrasound of kidneys, ureter and bladder) in 1:1 ratio between study and control groups will be recruited from among the healthy siblings of children presenting to the OPDs. Differential microRNA expression profiles in urine and serum samples of children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) with healthy children will be compared in a two-phased manner: a biomarker discovery phase involving pooled samples across SSNS, SRNS and healthy siblings analysed in triplicate using next-generation sequencing, slide microarray and quantitative reverse transcriptase PCR (qRT-PCR) arrays covering human miRNome followed by a validation phase with customised qRT-PCR primers based on the concordance in the discovery phase differential expression profiles and bioinformatics analysis. Ethics and dissemination:The study is funded after dueInstitutional Ethics Committee (IEC) clearance, and results will be available as open access.

Project description:The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Project description:ImportanceCalcineurin inhibitors are an established first-line corticosteroid-sparing therapy for patients with corticosteroid-dependent nephrotic syndrome (CDNS), whereas B-lymphocyte-depleting therapy is mostly used as a rescue for calcineurin inhibitor-resistant cases. The positive efficacy and safety profile of rituximab raises the question of whether it could be used as a first-line alternative to calcineurin inhibitor therapy.ObjectiveTo compare the efficacy of rituximab and tacrolimus in maintaining relapse-free survival among children with CDNS.Design, setting, and participantsA parallel-arm, open-label, randomized clinical trial was performed from May 8, 2015, to September 20, 2016, with 1-year follow-up in a single-center, tertiary care unit. A total of 176 consecutive children aged 3 to 16 years with CDNS not previously treated with corticosteroid-sparing agents were screened for eligibility.InterventionsThe children received either tacrolimus (along with tapering alternate-day prednisolone) for 12 months or a single course of rituximab (2 infusions of 375 mg/m2).Main outcomes and measuresTwelve-month relapse-free survival in the intention-to-treat population.ResultsOf the 176 children screened for eligibility, 120 were randomized and all but 3 patients completed 1 year of follow-up. The groups were comparable, with mean (SD) age of 7.2 (2.8) years, 32 boys (53.3%) in each group, mean (SD) disease duration of 2.5 (1.5) years and 2.3 (1.7) in the tacrolimus and rituximab groups, respectively, disease duration less than 1 year among 15 children (25.0%) in each group, median (interquartile range) of 4 (3-5) relapses in each group, and mean (SD) cumulative prednisolone dose of 246 (48) mg/kg and 239 (52) mg/kg in the prestudy year in the tacrolimus and rituximab groups, respectively. Rituximab therapy was associated with a higher 12-month relapse-free survival rate than tacrolimus (54 [90.0%] vs 38 [63.3%] children; P < .001; odds ratio, 5.21; 95% CI, 1.93-14.07). Among the patients who experienced relapse, median time to first relapse was 40 weeks in the rituximab group and 29 weeks in the tacrolimus group. Only 2 patients in the rituximab group had more than 1 relapse during the study period compared with 10 patients in the tacrolimus group. The cumulative corticosteroid dose during the 12-month study period was lower with rituximab compared with tacrolimus (mean [SD], 25.8 [27.8] vs 86.3 [58.0] mg/kg). Although both treatments were well tolerated, mild to moderate infections were twice as common in the tacrolimus group (26 [43.3%] vs 13 [21.7%] events).Conclusions and relevanceIn children with CDNS, rituximab appears to be more effective than tacrolimus in maintaining disease remission and minimizing corticosteroid exposure and, given its good tolerability and lack of nephrotoxic effects, may be considered as first-line corticosteroid-sparing therapy.Trial registrationClinicalTrials.gov Identifier: NCT02438982; Clinical Trial Registry of India: CTRI/2014/01/004355.

Dataset Information

Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout.

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Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout.

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