Plasminogen Injures Glomerular and Proximal Regions in Kidney Organoids
Human embryonic-stem-cell-derived kidney organoids showed compartment-specific injury after plasminogen exposure. Amiloride inhibited uPA activity and restored some markers, while apoptosis remained elevated.
Modeling Injury along the Nephron
Plasminogen can appear in urine during proteinuric kidney disease, but its direct effects on different human kidney cell populations are difficult to separate. Researchers at Mount Sinai and the University of Pennsylvania used kidney organoids to compare glomerular, proximal tubular and distal tubular responses within the same three-dimensional model. They also tested whether inhibition of urokinase-type plasminogen activator, or uPA, changed the injury pattern.
The organoids were generated from H9 human embryonic stem cells. At differentiation day 21, cultures received 0.25 μM human plasminogen for 24 hours, with or without 10 μM amiloride. Confocal microscopy and automated image analysis identified compartments using podocalyxin, LTL and E-cadherin. Measurements were normalized against controls from the same differentiation batch, allowing tissue-marker changes and cellular density to be compared within each compartment.
Establishing Target Inhibition
The study first evaluated uPA activity using a fluorescence-based assay. Amiloride produced concentration-dependent inhibition, with a reported IC50 of 3.91 μM and a 95% confidence interval of 3.59–4.23 μM. That experiment established that the drug could inhibit the measured enzyme activity in the experimental setting before the tissue-injury comparisons were interpreted.
Biological and technical replication were kept distinct. The uPA experiment used three biological replicates with technical triplicates. The gene-expression experiment used six biological replicates, each pooling RNA from 8–12 organoids within one differentiation experiment. Those pooled samples support the transcript comparisons; individual organoids within a pooled sample do not represent additional donors or independent cell lines.
Unequal Responses across Kidney Compartments
Plasminogen reduced podocalyxin signal and cellular density in glomerular regions. Additional staining showed lower nephrin, podocin and WT1, supporting a broader change in podocyte-associated features. Proximal tubules lost LTL signal and megalin expression and showed increased KIM-1, a marker associated with tubular injury. Distal tubular marker expression and cellular density were relatively preserved over the same exposure period.
The injury pattern was also visible in apoptosis measurements. Cleaved caspase-3 increased in glomerular and proximal tubular regions, while the distal compartment showed no significant corresponding change. Measuring these regions separately revealed a distribution of susceptibility that would be obscured by reporting a single whole-organoid viability value.
Amiloride improved selected markers, including podocin and WT1, but the response was incomplete. Nephrin was not restored in the imaging comparison, proximal tubular KIM-1 induction persisted, and apoptosis remained elevated in affected regions. The same treatment could therefore produce marker recovery while leaving an important form of cellular injury unresolved.
Combining Imaging with Gene Expression
Bulk qPCR supported changes in podocyte and proximal tubular programs, including reduced expression of NPHS1, NPHS2, PDXL, WT1, HNF4, LRP2 and CUBN after plasminogen exposure. Some distal markers and SYNPO were not significantly altered. Amiloride partly or fully restored several affected transcripts, although the magnitude of that recovery varied across biological replicates.
The model brings several injury measurements together, but its exposure geometry differs from a functioning kidney. The organoids lack a perfused glomerular filtration barrier, and plasminogen reaches the tissue through the surrounding culture medium. All experiments also used one H9 genetic background. These conditions define the immediate use of the system: studying compartment-specific injury and pharmacological responses in a controlled human tissue model.
The pharmacological result is consequently most useful as a set of separate observations. uPA activity was inhibited, certain tissue markers improved, and apoptosis did not fall. Preserving those three outcomes gives subsequent mechanistic studies a clearer starting point than treating the experiment as a uniform protective response.