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Characterisation of a Mouse Kidney Cortical Collecting Duct Cell Line Suggests Plasticity Between Cell Types
Author(s) -
Assmus Adrienne M,
Mansley Morag K,
Mullins Linda J,
Peter Audrey,
Mullins John J
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.857.18
Subject(s) - aldosterone , amiloride , cell culture , epithelial sodium channel , immunostaining , renal sodium reabsorption , intercalated cell , chemistry , medicine , endocrinology , microbiology and biotechnology , sodium , biology , kidney , reabsorption , immunohistochemistry , genetics , organic chemistry
The kidney collecting duct is composed of principal (PC) and intercalated (IC) cells responsible for the reabsorption of water and sodium, and the acid‐base balance, respectively. The immortalized mCCD cl1 cell line is widely used as a model for PCs since cells respond to glucocorticoid (MR) and mineralocorticoid (MR) hormones, such as aldosterone, and give reproducible electrophysiological responses. The effect of culture media on sodium transport in the mCCD cl1 cell line was tested by electrophysiological methods and immunostaining. The cells were cultured on filters in two media: the original medium published by Gaeggeler et al .(2005), and an alternative medium using a different source of sodium selenite supplement (Gibco). The cells transepithelial voltage and resistance across monolayers of cells was measured using an epithelial volt‐ohm‐meter, allowing calculation of the equivalent short circuit current by Ohm's law. Basal currents recorded from cells cultured in the original medium measured −9.97μA/cm 2 , were amiloride‐sensitive and stimulated by ~2.6 fold following aldosterone treatment (−26.78μA/cm 2 ). Cells cultured in the alternative medium exhibited negligible current and no response to aldosterone was observed. Immunostaining was performed in the alternative media on the mCCD cl1 cells and on isolated subclones using antibodies against PC and IC markers: Aquaporin 2 (Aqp2) and the alpha subunit of the epithelial sodium channel (α‐ENaC) for PC, Connexin 30 (Cx30) and vacuolar H + ATPase (V‐ATPase) for IC. Both the original cell line and the clonal sub‐lines exhibited varied expression of Aqp2 and Cx30, with evidence of co‐expression in sub‐populations of cells. 41.8% of cells from the parental sub‐line express both PC and IC markers whilst 23.8 to 58.7% cells in the clonal sub‐lines express both PC and IC markers. Expression of both PC and IC markers was confirmed by PCR analyses, and RNA sequencing. Immunostaining for α‐ENaC revealed staining across the apical membrane of the cells when cultured in the original medium and perinuclear staining when cells were cultured in the alternative medium. These results show that the provenance of the culture media supplements has a direct consequence on the phenotype of the cells and correlates with their capacity for sodium transport. Secondly, mCCD cl1 cells can present characteristics of both principal and intercalated cells and may have the capacity to switch between the two phenotypes. Support or Funding Information British Heart Foundation Center of Research Excellence PhD Studentship