Investigating kidney disease using animal, cell and organoid models: the role of polyphosphates and bacterial lipopolysaccharides behind kidney injury

Kidney diseases of acute and chronic forms are a major cause of mortality and morbidity. While the pathological manifestations of different kidney injury types are well characterized, the molecular drivers and mechanisms are poorly understood, particularly when it comes to microbes. Inorganic polyphosphates are anionic polymers with pleiotropic effects which are ubiquitously expressed in prokaryotes and eukaryotes. Medium or long-chain polyphosphates may be released to circulation from platelets or pathogenic bacteria respectively, where they can synergize with immunostimulatory agents, such as bacterial lipopolysaccharides. In this thesis, the chain length dependent effects of polyphosphates and lipopolysaccharides are examined on cytokine signaling in human leukocytes in Study I, and on acute kidney injury development in mice in Study II. In Study III, the high-throughput culture and screening applicability of human stem cell derived kidney organoid technology is evaluated based on renal structure development rates. Study I showed that polyphosphates inhibit type I interferon signaling dose and chain length dependently in human leukocytes. They decreased lipopolysaccharide-induced interferon-β transcription and secretion, STAT1 phosphorylation, and interferon stimulated gene transcription. On the contrary, they augmented lipopolysaccharide-induced leukocytic secretion of cytokines IL1α, IL1β, IL4, IL5, IL10, and IFNγ. Study II found that intravenous administration of long-chain polyphosphates in mice induced a kidney injury phenotype with glomerular microthrombi characteristic of thrombotic microangiopathies, which were accompanied by slit diaphragm integrity loss and albuminuria. The combination of long-chain polyphosphates and lipopolysaccharide resulted in marked multifocal acute tubular necrosis in the cortex, as well as augmentation of lipopolysaccharide-induced pro-inflammatory cytokine levels. On the other hand, medium-chain polyphosphate did not have notable effects alone or when combined with lipopolysaccharide. In Study III we established four high-throughput kidney organoid culture approaches to enable more robust screening experiments, which can meet statistical requirements and better identify technical and biological sources of variation. Quantitative high-content screening analysis of renal structures showed that the culture approach, stem cell line, experimental replication, and initial cell number explained 35-77% of the variability when fitted into multiple linear models. In conclusion, Studies I and II demonstrated that polyphosphates have notable chain length dependent effects on cytokine signaling and thrombosis in vitro and in vivo, however the specific immunomodulatory mechanisms remain unknown. Bacterial long-chain polyphosphates can have a prothrombotic role and augment pro-inflammatory cytokine signaling in a manner characteristic of immunothrombosis. Our high-throughput kidney organoid approach, described in Study III, provides an easily adaptable microplate-based culture system for kidney organoids and highlights the benefits of multivariate techniques to better distinguish sources of technical and biological variation.