The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid‐base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10 and Tfcp2l1, have been knocked out (KO) (2, 3). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self‐immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (4). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Tfcp2l1 and Adam10 were knocked out in mCCDcl1 cells using CRISPR‐Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted and the knockout clones were verified using T7 endonuclease analysis, DNA sequencing and immunocytochemistry. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans‐epithelial resistance Rte, voltage Vte, calculated short‐circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 mA/cm2 vs −8.1±0.4 mA/cm2 for control) as well as aldosterone response, decreased by 95% ( fold change 0.19±0.03 down 3.70±0.11 for control). Both Tfcp2l1 KO clones failed to form a resistive cell monolayer, with a dramatic decrease in Rte compared to control (down to 0.10±0.02 kΩ.cm2) as well as a complete disappearance of baseline and epithelial sodium channel (ENaC) mediated amiloride‐sensitive current. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and, in combination with single cell RNA‐Seq data, will identify new pathways involved in regulating cell phenotype.Support or Funding InformationBritish Heart Foundation Centre for Research Excellence (BHF, RE/13/3/30183) Kidney Research UK (KRUK, RP/026/20180305)
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