Proximal tubule-enhanced kidney organoids with S3-like cell enrichment model physiological responses

Chronic kidney disease (CKD) is a major global health challenge affecting >840 million people, with rising prevalence driven by common conditions including diabetes, hypertension, obesity, and drug toxicity. The proximal tubule (PT) is central to kidney function yet is highly injury-prone, particularly within the S3 segment which plays key roles in CKD progression. Despite decades of in vitro PT model development, achieving accurate functional and structural segmentation of the PT with distinct PT sub-cell types has remained challenging. Here, we report an enrichment of S3-like cells within PT-enhanced kidney organoids (PT-EKO) that confers segment-specific functionality. This advanced platform facilitated modelling of physiological responses to environmental changes, including glucose levels, fatty acid substrate availability, and fluid sheer stress. Integrating with both static and organ-on-chip culture systems, the translational potential of S3-enriched PT-EKOs was underscored by their amenability to scale-up via cryopreservation of day 13 progenitors with retained differentiation capacity. PT-EKO applications were further broadened as an expandable high-yield source of S3-like PT cells, retaining transporter functionality across multiple passages. Together, these findings represent an advance towards more physiologically relevant, human-specific modelling of PT responses supporting therapeutic approaches for CKD, including drug development, toxicity evaluation and cellular therapy.