Kidney organoids as a platform to study plasminogen-induced injury and uPA-based therapies.

Abstract

Urinary plasminogen has been implicated in the progression of proteinuric kidney diseases, yet its direct role in mediating injury along the nephron remains incompletely understood. Experimental models commonly used to study plasminogen-mediated damage are limited by incomplete phenotypic fidelity or interspecies differences, hindering translational relevance. Here, we used kidney organoids generated from H9 human embryonic stem cells to investigate the effects of plasminogen exposure and to evaluate urokinase-type plasminogen activator (uPA) inhibition with amiloride as a therapeutic strategy. Kidney organoids were exposed to plasminogen in the presence or absence of amiloride, and uPA activity was quantified using a fluorescence-based assay. Compartment-specific injury was assessed by confocal microscopy, automated image analysis, and AI-assisted nuclear segmentation, while targeted transcriptomic changes were evaluated using bulk quantitative PCR. Plasminogen exposure induced glomerular injury, characterized by reduced podocalyxin, nephrin, podocin, and WT1 expression, decreased glomerular cell density, and increased apoptosis. Proximal tubules exhibited reduced LTL and megalin expression, with increased KIM-1, whereas distal tubules were relatively preserved. Amiloride effectively inhibited uPA activity and partially restored podocyte and proximal tubular marker expression, but did not prevent apoptosis, indicating incomplete protection against plasminogen-induced injury. These findings support preferential plasminogen-induced injury along the glomerular-proximal tubular axis and highlight the utility of human kidney organoids for modeling this process, while underscoring the need for further validation and broader molecular profiling to strengthen their application in mechanistic studies and therapeutic discovery.