Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

Abstract

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Keywords: 2D iPSC-derived podocytes; Human iPSC-derived kidney organoids; Nephrotic syndrome.

Fig. 1.

Human iPSC-derived kidney organoids contain 17 distinct cell clusters, including well-developed podocytes showing filtration slits. (A) Schematic of the iPSC-derived kidney organoid culture protocol. iPSCs were differentiated in 2D either towards anterior intermediate mesoderm (AIM) or posterior intermediate mesoderm (PIM) and aggregates (300,000 cells) were made using a 1:2 AIM/PIM ratio on day 7. Organoids were cultured on the air-liquid interface of Transwell™ filters for an additional 18 days, using a mixture of growth factors as indicated. BMP7, bone morphogenic protein 7; CHIR, CHIR 99021; EGF, epidermal growth factor; FGF9, fibroblast growth factor 9. (B) Uniform manifold approximation and projection (UMAP) integration of 3900 single cells from human iPSC-derived kidney organoids results in 17 different cell clusters. CD, collecting duct precursors; DT, distal tubular cells; ECp, endothelial cell precursors; LH, Loop of Henle cells; NP, neuronal progenitors; PT, proximal tubular cells. (C-C″) Segmented patterning in human kidney organoids shows the presence of podocytes (NPHS1, magenta), proximal tubules [lotus tetragonolobus lectin (LTL), blue], distal-like [E-cadherin (ECAD), green] and collecting duct-like [ECAD (green) and GATA binding protein 3 (GATA3) (red)] cells. (D-E′) TEM micrographs of organoid-derived podocytes showing cell bodies and interdigitating foot processes including a filtration slit, indicated by an arrowhead in E′. (F-H) Filtration slit proteins podocin (red) and nephrin (green) are expressed at the podocyte cell membranes. (I-K) The podocyte-specific transcription factor Wilms' Tumor 1 (WT1, green) is expressed in podocyte nuclei (podocin, red; nuclei, blue). (L-N) Phospholipase A2 receptor (PLA2R, red) expression at the podocyte cell membranes (nephrin, green). (O-P′) This cluster was subsetted and merged with the subsetted podocytes from the organoids. Super-resolution filtration slit imaging of nephrin in organoid-podocytes (O,O′) and in a morphologically healthy appearing glomerulus of a Munich Wistar Frömter rat (P,P′).

Fig. 2.

Kidney organoid-derived podocyte subcluster 3 partially resembles adult podocytes. (A,B) UMAP integration of 401 podocytes (A) identified three subclusters based on differentially expressed genes (B). Avg, average; Pct, percent. (C) RNAscope analysis showing nephrin (NPHS1), collagen IV alpha three (COL4A3) and endothelial marker cluster of differentiation 31 (CD31) expression. Inset shows an enlarged view of the boxed area. (D) Vascular endothelial growth factor A (VEGFA) expression among the podocyte subclusters. (E,F) CD31-, VE-cadherin- and plasmalemmal vesicle associated protein-1 (PV-1)-stained endothelial capillaries are interspaced between podocytes expressing NPHS1 (E) and synaptopodin (SYNPO) (F) in the organoids. Arrowheads indicate endothelial capillaries with lumen. (G) Ultrastructural analysis showing the luminal space of a capillary adjacent to organoid podocytes. (H) Cell-cell interactions between all cells present in kidney organoids using CellPhoneDB and CrossTalkeR packages. Thickness and depth of color of the arrows correspond to the percentage of interactions. (I) Autocrine and paracrine podocyte ligand-receptor interactions showing a dominant role for VEGFA signaling originating from cluster 3 to VEGF-related receptors in, amongst others, endothelial cell precursors (EC). Analysis was performed using CellPhoneDB and CrossTalkeR packages. (J-L) Comparative PROGENy (J), GO terms (K) and DoRothEA analysis (L) showing pathway activity (J), biological processes (K) and transcription factor activity (L) between the three podocyte subclusters, adult and fetal podocytes [adult and fetal podocytes sequencing data extracted from Gene Expression Omnibus (GSE118184 and GSE112570)]. Abbreviations as in Fig. 1.

Fig. 3.

Organoid podocytes show superior morphology and transcriptome compared with 2D podocytes in vitro. (A-C) TEM micrographs showing the ultrastructural analysis of normal human podocytes in situ (A), conditionally immortalized podocytes (ciPOD) (B) and organoid podocytes (C). (D) Heatmap of bulk RNA sequencing data showing the expression signature of podocyte-specific markers in normal human glomeruli, ciPODs and organoid podocytes.

Fig. 4.

Congenital nephrotic syndrome modeling of patient-derived NPHS2 mutant and genetically repaired iPSC kidney organoids. (A-F) Nephrin (magenta) and podocin (green) expression in NPHS2 mutant (A-C) and genetically repaired (D-F) kidney organoids. Insets show enlarged views of the boxed areas. (G) Comparative bulk transcriptomics of podocyte-specific genes in mutant versus repaired organoid podocytes. (H,I) Comparative PROGENy pathway activity analysis (H) and DoRothEA transcription factor activity (I) of mutant versus repaired organoid podocytes. (G-I) Bulk transcriptomics was performed from RNA isolated from three independent batches of NPHS1⁺ sorted podocytes from mutant and repaired organoids. Per batch, ten mutant or repaired organoids were pooled and used for sorting podocytes by FACS.

Fig. 5.

Kidney organoids are the preferred model of choice to model idiopathic nephrotic syndrome. (A-H) Podocyte cytoskeleton [phalloidin (green), NPHS1 (magenta)] (A-D) and ultrastructure analysis (E-H) of protamine sulfate-treated and heparin-rescued kidney organoid podocytes. (I-L) Podocyte cytoskeleton analysis [phalloidin (green), NPHS1 (magenta)] of protamine sulfate-treated and heparin-rescued 2D iPSC-derived podocytes. (M-P) Podocyte cytoskeleton analysis [phalloidin (green) and NPHS1 (magenta)] of protamine sulfate-treated and heparin-rescued 2D ciPODs. (Q) Quantification of phalloidin intensity in protamine sulfate-treated and heparin-rescued kidney organoid podocytes. *P<0.05, **P<0.01 (one-way ANOVA analysis followed by Tukey post-test). Data are presented as mean±s.d. from three independent experiments using at least two biological replicates per condition. (R) Western blotting analysis of pNPHS1 and NPHS1 protein expression in protamine sulfate (PS)-treated kidney organoids and 2D iPSC-derived podocytes. Gamma tubulin was used as a reference protein. (S) Semi-quantification of the pNPHS1/NPHS1 ratio of protamine sulfate-treated kidney organoids. *P<0.05 (unpaired t-test). Data are presented as mean±s.d. from three independent experiments using at least two biological replicates per condition.

Fig. 6.

Puromycin aminonucleoside-induced injury in kidney organoids results in autophagic activity comparable to human minimal change disease NS. (A) Ultrastructural analysis of a human MCD kidney biopsy specimen as observed by TEM. The podocyte (dashed line, P) showed vacuole formation (examples marked with asterisks), foot process effacement (FPE) and autophagosomes (AP). N, nucleus. (B) 3D volume rendering of vehicle-treated kidney organoid, imaged by FIB-SEM, showing podocytes (yellow, magenta) and foot processes. The complete z-stack, including annotations, is shown in Movie 1. (C) 3D rendering of the segmented podocytes shows interdigitating podocyte foot processes (arrows). See Movie 2 for 3D rendering following segmentation. (D-F) FIB-SEM volume imaging (stack slide numbers: D, 327; E, 735; F, 1456) from PAN-injured podocytes (P) showing autophagosomes (APs), lysosomes (L), foot processes (FP) with ruptured membranes (RM). AL+G, autolysosome with glycogen; M, mitochondria; N, nucleus; T, tubular cells. Scale bars: 2 µm. The complete z-stack, including annotations, is shown in Movie 3.