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Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development

ABSTRACT: A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+) and microvillus-rich apical membranes (podocalyxin+), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103547 | GEO | 2017/09/20

SECONDARY ACCESSION(S): PRJNA401729

REPOSITORIES: GEO

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Project description:Objectives: Secretion of extracellular vesicles (EV) and associated micro-RNAs (miR) is altered during cellular stress and may serve as biomarkers of organ injury. We hypothesized that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. Design: Predictive accuracy biomarker study performed in the cohort of the REVAKI-2 trial Setting: Single center ICU between September 2015 and September 2018 Interventions: Intravenous sildenafil citrate 12.5 mg kg-1 over 150 min or dextrose 5% at the commencement of surgery. Measurements and main results: Urine samples were collected before and 24 hours after the procedure from 93 cardiac surgery patients. Urine EV concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced to detect differentially expressed miR. Verification was performed with advanced TaqMan assays in samples from all patients. Urine EV concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for aquaporin-2 and β1-integrin also increasing. Pre surgery podocalyxin-positive EV were significantly lower, and β1-integrin EV werehigher in patients with AKI. The levels of the former correlated with the severity of theinjury. miR-125a-5p was expressed at higher levels in urine from patients with AKI stage 2/3. Levels of miR-10a-5p decreased after surgery in AKI patients; its levels correlated with the severity of the injury. Preoperative levels of podocalyxin EVs and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 80.9%) AKI prediction. Conclusions: Lower levels of podocalyxin-positive EV at baseline predict the severity of post-surgery AKI. Urine EV concentrations and miR expression offer excellent predictive accuracy when combined with commonly measured biomarkers.

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Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development

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