Abstract
ABSTRACTCystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.
Highlights
In vitro models of stem cell differentiation and kidney development have set the stage for expanding our understanding of nephrogenesis
This disconnect is in part due to the dramatic change in morphometry and organization of epithelial cells on flat, stiff substrates (Fig. 1A)
We hypothesized that morphological changes precede alterations to proliferation and secretion after polycystin loss
Summary
In vitro models of stem cell differentiation and kidney development have set the stage for expanding our understanding of nephrogenesis. Optimized variations of 3D epithelial culture using induced pluripotent (iPSC) and adult (ASC) stem cells have led to novel kidney model systems that are able to recapitulate intermediate mesoderm, metanephric mesenchyme, ureteric epithelium, and proximal tubules (Freedman et al, 2015; Takasato et al, 2016; Schutgens et al, 2019). Introduction of specific mutations by CRISPR-Cas gene editing can be used to model human diseases of the kidney (Clevers, 2016; Freedman et al, 2015). These intricate models remain unmatched in recapitulating the complexity of the developing kidney. Organoid complexity is not always a desirable feature, especially for studies focused on identifying and characterizing molecular pathways that drive tubulogenesis, epithelial cell differentiation, and complex disease processes, such as autosomal dominant polycystic kidney disease (ADPKD) (Clevers, 2016)
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