Pirttiniemi, A. | Search bioRxiv

bioRxiv2025

Long-chain polyphosphates induce glomerular microthrombi and exacerbate LPS-induced acute kidney injury in mouse

Polyphosphates are evolutionarily conserved anionic polymers mediating pleiotropic functions in eukaryotes and prokaryotes, depending on their chain-length. Bacteria typically synthetize long-chains, while human platelets harbour exclusively medium-chains. Polyphosphate-mediated lung and liver-injury have been reported in experimental mouse models but their effects on the kidney remain undefined. Here we assessed kidney histopathology and cytokine levels following intravenous administration of medium-chain (P100) and long-chain (P700) polyphosphates and their synergistic effects with lipopolysaccharides (LPS) in mice. We found that P700 induced albuminuria, renal Kim-1 and Lcn2 transcription, focal renal damage with glomerular microthrombi, tubular degeneration, granular phenotype of slit diaphragm components nephrin and ZO1, and enlarged electron dense vesicles in podocyte cytoplasm indicating lysosome swelling. P700 combined with LPS induced marked multifocal acute tubular necrosis in the cortex, and augmented LPS-induced proinflammatory cytokine levels. No notable effects were seen with P100, indicating that PolyP-mediated kidney injury development is dependent on chain-length. We conclude that long-chain polyphosphates may play a procoagulant role behind kidney injury, by inducing microthrombi characteristic of thrombotic microangiopathy and augmenting cytokine levels under inflammatory conditions.

pathology↗

bioRxiv2025

Parsing glomerular and tubular structure variability in high-throughput kidney organoid culture

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35-77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguishing sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability.

developmental biology↗