Open AccessLoss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells
Author(s) -
Adrienne Assmus,
L. J. Mullins,
Mairi Ward,
Ross Dobie,
Robert W. Hunter,
Neil Henderson,
John J. Mullins
Publication year - 2021
Publication title -
function
Language(s) - English
Resource type - Journals
ISSN - 2633-8823
DOI - 10.1093/function/zqab024
Subject(s) - intercalated cell , epithelial sodium channel , biology , gene knockdown , microbiology and biotechnology , immunostaining , knockout mouse , kidney , hek 293 cells , cell culture , cell type , cell , chemistry , endocrinology , sodium , immunology , genetics , immunohistochemistry , gene , organic chemistry
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, mCCD cl1 , 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 mCCD cl1 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.