Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics
Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics
ABSTRACT BackgroundDNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls. ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease. SIGNIFICANCE STATEMENTGaps persist in our mechanistic understanding of APOL1-mediated kidney disease. The authors apply genome-edited human kidney organoids, combined with single-cell transcriptomics, to profile APOL1 risk variants at the native genomic locus in different cell types. This approach captures interferon-mediated induction of APOL1 gene expression and reveals cellular dedifferentiation after a secondary insult of endoplasmic reticulum stress. This system provides a human cellular platform to interrogate complex mechanisms and human-specific regulators underlying APOL1-mediated kidney disease.
Liu Esther、Donnan Michael D.、Smith Kelly D.、Himmelfarb Jonathan、Lin Jennie、Radmanesh Behram、Dadi Amal、Yi Dan、Freedman Benjamin S.、Chung Byungha H.、Li Mingyao
Division of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of MedicineDivision of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of MedicineDepartment of Pathology, University of WashingtonDivision of Nephrology, Kidney Research Institute, and Institute for Stem Cell and Regenerative Medicine, Department of Medicine, University of WashingtonDivision of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of Medicine||Section of Nephrology, Jesse Brown Veterans Affairs Medical CenterDivision of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of MedicineDivision of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of MedicineDivision of Nephrology and Hypertension, Feinberg Cardiovascular and Renal Research Institute, Department of Medicine, Northwestern University Feinberg School of MedicineDivision of Nephrology, Kidney Research Institute, and Institute for Stem Cell and Regenerative Medicine, Department of Medicine, University of Washington||Department of Pathology, University of WashingtonDivision of Nephrology, Kidney Research Institute, and Institute for Stem Cell and Regenerative Medicine, Department of Medicine, University of WashingtonDepartment of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania
基础医学生物科学研究方法、生物科学研究技术分子生物学
induced pluripotent stem cellsgenome editingnephron progenitor cellsfunctional genomicsfocal and segmental glomerulosclerosisendoplasmic reticulum stress
Liu Esther,Donnan Michael D.,Smith Kelly D.,Himmelfarb Jonathan,Lin Jennie,Radmanesh Behram,Dadi Amal,Yi Dan,Freedman Benjamin S.,Chung Byungha H.,Li Mingyao.Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics[EB/OL].(2025-03-28)[2025-06-15].https://www.biorxiv.org/content/10.1101/780684.点此复制
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