A Novel Human Distal Tubuloid-On-A-Chip Model For Investigating Sodium And Water Transport Mechanisms

The distal segments of the nephron play a central role in regulating water and electrolyte balance, making them critical targets for therapeutic interventions. Damage to these segments is associated with significant health consequences. Studying their (patho)physiology in vivo remains challenging due to the kidney's complex architecture. Recent advances led to the development of more representative in vitro models, including human tubuloids that replicate the phenotype of distal tubule segments. Additionally, novel high throughput microfluidic systems, which support 3D cell culture under flow conditions, provide a platform for closely mimicking in vivo environments. This study presents an enhanced in vitro model of human distal tubule segments by integrating tubuloid culture with the OrganoPlate platform. Tubuloid cells were grown as three-dimensional tubules against a collagen-1 matrix and under alternating flow condition, then differentiated into a distal phenotype. qPCR analysis demonstrated enhanced expression of distal segment markers in 3D flow cultures compared to traditional 2D models. Immunohistochemistry confirmed the formation of a leak-tight, highly polarized epithelium with apical and basolateral localization of key electrolyte transporters. Functional integrity was verified by restricted dextran diffusion and increased transepithelial resistance. Radiolabeled sodium assays revealed active and selective sodium transport mediated by apical epithelial sodium channels (ENaC) and basolateral Na/K ATPase. Sodium transport was followed by water movement, evidenced by dome formation beneath the epithelial monolayer. The model's utility was further demonstrated in toxicity studies using trimethoprim, an antibiotic that inhibits ENaC function, resulting in reduced sodium transport and dome formation. This system enables the study of primary human tubule cells with a distal phenotype under controlled flow conditions, allowing direct assessment of water and salt transport. The model provides a valuable tool for investigating distal nephron (patho)physiology and facilitates high-throughput drug development and toxicity testing.