Loss of Adam10 disrupts ion transport in immortalised kidney collecting duct cells
Data CreatorMullins, John
PublisherUniversity of Edinburgh; BHF Centre for Cardiovascular Science
MetadataShow full item record
CitationMullins, John; Assmus, Adrienne; Mullins, Linda. (2021). Loss of Adam10 disrupts ion transport in immortalised kidney collecting duct cells, [dataset]. University of Edinburgh; BHF Centre for Cardiovascular Science. https://doi.org/10.7488/ds/3022.
DescriptionThe kidney cortical collecting duct (CCD) comprises of principal cells (PC), intercalated cells (IC) and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess (SAME) revealed plasticity of the cortical collecting duct (CCD), with altered principal cell (PC): intermediate cell: intercalated cell (IC) ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCDcl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (gamma-ENaC) and Hsd11b2 (11ßHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of gamma-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarisation.
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