Fig. 6

Organoid Glomeruli model of congenital nephrotic syndrome in vitro. a Description of the NPHS1 variants identified in the patient modelled, diagnosed with congenital nephrotic syndrome (CNS). b–d Immunostaining of OrgGloms isolated from control organoids and CNS patient organoids show reduced NEPHRIN and PODOCIN protein levels in the organoids derived from patient-iPSC, representative images shown of >3 biological replicates. Scale bars 10 µm. e Higher power immunofluorescent images show the polarised co-localisation of NEPHRIN with NEPH1 (solid white arrowheads) and PODOCIN in control OrgGloms. This polarisation is lost in CNS OrgGloms due to the absence of NEPHRIN (white arrows). Scale bars 10 µm. f Quantitative analysis of fluorescence intensities from independent OrgGlom biological replicates performed using one control and two distinct patient-derived CNS iPSC clones. Organoid glomeruli generated from both patient-derived iPSC clones show significant reduction in NEPHRIN and PODOCIN protein levels. Two-way ANOVA p < 0.0001; error bars = SEM. Biological replicates. NEPHRIN (controls, n = 20; CNS, n = 56); PODOCIN (controls, n = 14; CNS, n = 22); CD2AP (controls, n = 8; CNS, n = 15); NEPH1 (controls, n = 10; CNS, n = 17). Significant difference assessed by Sidak’s multiple comparisons test between cell lines; F-value = 112; DF = 1. NEPHRIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p > 0.9999. PODOCIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p = 0.9995. CD2AP: control vs CNS#1, p = 0.0007; control vs CNS#2, p = 0.0016; CNS#1 vs CNS#2, p = 0.9980. NEPH1: control vs CNS#1, p = 0.5320; control vs CNS#2, p = 0.9994; CNS#1 vs CNS#2, p = 0.9992. g Quantitative western blot analysis of NEPHRIN and PODOCIN protein levels within independent biological replicates confirms the significant depletion of these proteins in OrgGloms derived from CNS iPSCs