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 heads towards adult podocytes and was superior compared to 2D counterparts, based on scRNA 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.

Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids

“…Similar to tubuloids, podocytes disappear in iPSCod tubuloids over the first passages, which probably reflects the poor proliferative potential of these cells in vivo ( 55 , 56 ). Therefore, organoids from iPSCs are the current model of choice for studies of glomerular disease ( 10 , 18 , 50 , 57 ).…”

“…For applications that require these compartments, combination with other approaches (e.g., isolation of these cell populations from cell lines, primary tissue or regular iPSC organoids) is probably required. Finally, we confined our analysis to iPSCod tubuloids established from organoids that were grown using one protocol in the field ( 1 ), whereas many different protocols are available that give rise to renal organoids with different cell compositions ( 2 , 12 , 18 , 48 – 50 , 58 , 59 ). In the future, the differences between iPSCod tubuloids established from different protocols should be compared, in particular for protocols that further mature or enrich for specific cell types ( 12 , 48 , 49 ).…”

“…The renal precursor tissues then exchange reciprocal endogenous signals to autonomously form podocytes, tubular epithelium, vasculature, and stromal cells organized in nephron-like structures ( 1 , 2 , 6 – 8 ). Human kidney organoids have been used for a variety of purposes, including studies of organ development, disease modeling, drug efficacy and toxicity testing, and first steps toward regenerative medicine ( 8 – 18 ). These applications have been facilitated by introduction of knockouts and reporters ( 9 , 11 , 14 , 19 – 21 ).…”

Protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell-derived kidney organoids using Seahorse XF96

“…Mature kidney organoids show the presence of podocytes (Nephrin, NPHS1), proximal tubules (lotus tetragonolobus lectin, LTL), distal-like (E-cadherin, ECAD) and collecting duct-like (ECAD and binding protein 3 (GATA3)) cells [ troubleshooting 3 ]. 4 , 5 , 10 , 11 Note: If treatments are needed to induce kidney organoid injury, they should be applied after completion of morphogenesis both on top and below the Transwell filter with 800 μL and 1.2 mL of E6 medium, respectively, to gain maximum exposure surface area. Treatments have to be maintained after re-plating into Seahorse microplates.…”

“…Mature kidney organoids show the presence of podocytes (Nephrin, NPHS1), proximal tubules (lotus tetragonolobus lectin, LTL), distal-like (E-cadherin, ECAD) and collecting duct-like (ECAD and binding protein 3 (GATA3)) cells [ troubleshooting 3 ]. 4 , 5 , 10 , 11 …”