Characterisation of a Mouse Kidney Cortical Collecting Duct Cell Line Suggests Plasticity Between Cell Types

Adrienne Assmus, Morag Mansley, Linda Mullins, Audrey Peter, John Mullins

Research output: Contribution to conferenceAbstractpeer-review

Abstract

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 mCCDcl1 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 mCCDcl1 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/cm2, were amiloride-sensitive and stimulated by ~2.6 fold following aldosterone treatment (−26.78μA/cm2). 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 mCCDcl1 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, mCCDcl1 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
Original languageEnglish
Publication statusPublished - Apr 2017
EventExperimental Biology 2017 - Chicago, United States
Duration: 22 Apr 201726 Apr 2017
http://experimentalbiology.org/2017/home.aspx

Conference

ConferenceExperimental Biology 2017
CountryUnited States
CityChicago
Period22/04/1726/04/17
Internet address

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