mCCD_cl1cells show plasticity consistent with the ability to transition between principal and intercalated cells

Abstract

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCD cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCD parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCD cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCD cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCD cells being precursor cells.

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells

“…Immunostaining of markers specific to PCs and ICs, Aqp2 and V-ATPase B1, respectively, shows altered expression levels and localization of proteins. Parental mCCD cl1 cells showed varied expression levels in individual cells, and a staining distribution in line with previous report 19 : ∼44% of cells with no staining; ∼41% with dual staining of Aqp2 and V-ATPase B1; and a small proportion of cells staining for only Aqp2 (8.7%) or V-ATPase B1 (5.3%). In contrast, A1 showed an overall decrease in Aqp2 expression (−41.4% ± 3.7% in A1) but also a more uniform expression of both Aqp2 and V-ATPase B1 across the whole cell population ( Figure 7A, C, and D ).…”

“…Despite including a significant portion of cells with an intermediate phenotype 19 (expressing both PC and IC markers), mCCD cl1 cells exhibit the expected functions of PCs such as amiloride-sensitive sodium transport. It is not clear whether intermediate cells in vitro (or in vivo) are capable of some physiological function.…”

“…The intermediate cells are phenotypically similar to those found in vivo, indicating a capacity for plasticity. 19 This makes them an ideal system for gene targeting, using CRISPR/Cas9, to interrogate the phenomenon.…”

Novel Human Kidney Cell Subsets Identified by Mux-Seq

“…More than 11,000 human collecting duct cells have been analyzed in this study, and transitional cells are noted in the human collecting duct, consisting mostly of PC cell type that can transition into IC cells. The presence of transitional cells in collecting duct in rodents has been described before 15,50 .…”

Cellular plasticity: A mechanism for homeostasis in the kidney

“…In vitro models of collecting duct cells such as mCCD cl1 cells (self‐immortalized mouse CCD cell line) have shown transitional phenotype too, and capacity for plasticity retained through single cell cloning . Interestingly, mCCD cl1 cells still exhibit the expected functions of PCs such as amiloride‐sensitive sodium transport, suggesting that hybrid cells are still capable of physiological function, but that observation has not been confirmed in vivo.…”