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Abstract Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

The use of differentiating human induced pluripotent stem cells (hiPSCs) in mini-tissue organoids provides an invaluable resource for regenerative medicine applications, particularly in the field of disease modeling. However, most studies using a kidney organoid model, focused solely on the transcriptomics and did not explore mechanisms of regulating kidney organoids related to metabolic effects and maturational phenotype. Here, we applied metabolomics coupled with transcriptomics to investigate the metabolic dynamics and function during kidney organoid differentiation. Not only did we validate the dominant metabolic alteration from glycolysis to oxidative phosphorylation in the iPSC differentiation process but we also showed that glycine, serine, and threonine metabolism had a regulatory role during kidney organoid formation and lineage maturation. Notably, serine had a role in regulating S-adenosylmethionine (SAM) to facilitate kidney organoid formation by altering DNA methylation. Our data revealed that analysis of metabolic characterization broadens our ability to understand phenotype regulation. The utilization of this comparative omics approach, in studying kidney organoid formation, can aid in deciphering unique knowledge about the biological and physiological processes involved in organoid-based disease modeling or drug screening.

Summary: Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function.For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

Summary: Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development. : In this study, Przepiorski et al. present a technically simple and robust protocol to derive kidney organoids from human induced pluripotent stem cells. This technique is inexpensive and efficient, allowing kidney organoids to be generated in bulk. Organoids are obtained rapidly (within 8 days) and, based on their segmentation pattern, correspond to “late capillary loop” stage fetal human nephrons. Keywords: iPSC, kidney organoid, bioreactor, HNF1B, renal development, CRISPR/Cas9, 3D culture, embryoid body, fetal human kidney, fibrosis