Abstract
The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate 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 (γ-ENaC) and Hsd11b2 (11βHSD2), 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 γ-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 polarization.