Maturation-enhanced proximal tubules enable functionality, toxicity screening, and infectious disease modeling in kidney organoids

ABSTRACT

Background: The highly specialised proximal tubule (PT) nephron segment is responsible for most kidney functions in mammals and is acutely vulnerable to disease, making it a key objective for toxicity screening and disease research. While induced pluripotent stem cell (iPSC)-derived kidney organoids represent a promising approach, the PT remains immature with limited evidence of functional transporters. Here we report the development of PT-enhanced organoids with nephron functionality, enabling improved modelling of PT-relevant conditions including drug-induced toxicity and infection such as SARS-CoV-2. Methods: Standard and fluorescent reporter iPSC lines were subjected to prolonged monolayer differentiations (modified from Howden et al. EMBO Rep 2019;Vanslambrouck et al. JASN 2019) and precisely-timed morphogens for targets such as WNT, BMP and NOTCH pathways prior to organoid generation (Takasato et al,. Nat Protoc 2016). Maturation was analysed via immunofluorescence, live confocal imaging of fluorescent reporters, transcriptional profiling, transporter function assays, and SARSCoV-2 infectivity. Results: Prolonged nephron progenitor differentiation with simultaneous prevention of spontaneous nephrogenesis resulted in PT-enhanced kidney organoids with elongated and aligned nephrons. Striking proximo-distal nephron orientation resulted from localised WNT antagonism. Improved upregulation of PT-specific markers compared to standard organoids was strengthened by evidence of transporter functionality, including uptake of albumin, organic cations, and cisplatin (eliciting appropriate KIM1 upregulation). This approach also improved expression of SARS-CoV-2 entry factors, confirmed by susceptibility to infection and viral replication. Conclusions: We describe enhanced kidney organoids with improved PT maturity arising from alterations to early mesodermal patterning and delayed nephron initiation. The enhanced conditions also provided more stringent control over nephron spatial arrangement. PT-enhanced organoids provide an ideal model to better understand human PT maturation, inherited and acquired PT disease, and drug toxicity implications.