Fig 2: Expression of kidney markers (SIX2, PAX8, Nephrin, and Podocin), endothelial marker (CD31), mesenchymal markers (PDGFR-ß and Vimentin), and pluripotency-associated markers (TRA-1-81, NANOG, and SSEA4) in 3D-NPCs. Cell nuclei were stained with Hoechst. Zoom-in pictures show the subcellular localization of the respective proteins. Pictures were taken under 20x magnification. The scale bars represent a length of 50 µm.

Fig 3: Reduced expression of podocyte-specific proteins in a PAN injury model is reversible in vitro.Localization and expression of podocyte-specific proteins NPHS1, NPHS2, CD2AP, and SYNPO (red) and F-actin (green) in differentiated, PAN (25 µg/ml)–treated LY podocytes and 5-day PAN washout of LY podocytes assessed by immunocytochemistry. DAPI (blue) detects nuclei. Scale bar, 50 µm.

Fig 4: Correlation of GOAC proteinuria with clinical data and MN mechanism modeling on the chip. a Table of clinical parameters for proteinuria in MN serum samples used on the GOAC. b hAKPC-P + hGEC chip proteinuria for CTRL1-2 and MN1–6 clinical proteinuria levels suggests a very strong correlation between clinical profile and response in the chip (measured as albumin leakage). R: 0.8901, P < 0.01. c hiPOD + hGEC chip proteinuria for CTRL1–2 and MN1–6 clinical proteinuria levels suggests a weak correlation between clinical profile and response in the chip (measured as albumin leakage). R: 0.3156, not significant. d hpPOD + hGEC chip proteinuria for CTRL1–2 and MN1–6 clinical proteinuria levels suggests a strong correlation between clinical profile and response in the chip (measured as albumin leakage). R: 0.7995, p < 0.05. For all samples, regression analysis was performed. Equation: Polynomial, linear. Blue lines = 95% confidence band; red lines = 95% prediction band. e, f Western blot analysis for C3d (140kDA) and beta actin (40 kDa) in hAKPC-P + hGEC, hiPOD + hGEC, and hpPOD + hGEC chips exposed to healthy or MN serum confirmed increased expression for C3d by all three MN chips. Number of replicates per experimental group: 3 (f). Quantification of C3d expression was performed by measuring pixel density and followed by normalization against beta actin. g, h Western blot analysis for NPHS1 (138kDA) and beta actin (40 kDa) in hAKPC-P + hGEC, hiPOD + hGEC, and hpPOD + hGEC chips exposed to healthy or MN serum confirmed decreased expression for NPHS1 by all three MN chips. Number of replicates per experimental group: 3. h Quantification of NPHS1 expression was performed by measuring pixel density and followed by normalization against beta actin (g). For all samples lack of significant differences was determined by a one-way ANOVA and Student–Newman–Keuls post hoc test. Box plots show the median, the 25th and 75th percentiles, whiskers (median ± 1.5 times interquartile range), and outliers (solid circle). Western blot images were cropped to show the relevant bands and improve clarity. Uncropped and unprocessed scans for western blotting analysis are provided in Supplementary Figs. 13, 14

Fig 5: Differentiation of LY podocytes.(A) Phase-contrast microscopy of proliferating LY human podocytes cultured at 33°C. Differentiation of LY podocytes at 37°C for 12 days results in a transition to differentiated morphology. Differentiated cells increase in size up to lengths of 150 µm and develop finger-like foot processes around the periphery of the cell. Scale bar, 50 µm. (B) Scanning electron microscopy reveals ultrastructures of primary and secondary foot processes on differentiated LY podocytes. (I) LY podocyte differentiated at 37°C for 12 days demonstrates primary foot processes around the cell periphery and extending toward adjacent cell. Scale bar, 20 µm. (II) Primary foot processes interdigitating between differentiated LY podocytes. Scale bar, 5 µm. (III) Primary foot processes branching off of a differentiated LY podocyte. Scale bar, 5 µm. (IV) Secondary foot processes around the outer periphery of a differentiated podocyte. Scale bar, 10 µm. (C) Localization and expression of podocyte-specific proteins NPHS1, NPHS2, CD2AP, and SYNPO (red) in proliferating and differentiated LY podocytes assessed by immunocytochemistry at low magnification. DAPI (4',6-diamidino-2-phenylindole) (blue) detects nuclei. Scale bar, 200 µm. (D) Localization and expression of podocyte-specific proteins NPHS1, NPHS2, CD2AP, and SYNPO (red) in a single differentiated LY podocyte. DAPI (blue) detects nuclei. Scale bar, 50 µm; right panels, magnification of area marked by white boxes.