Aldosterone‐Induced Transcripts Identified from Rapidly Isolated Collecting Duct Cells

Abstract

In the distal nephron, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel ENaC. Previous attempts to identify the aldosterone‐induced proteins that mediate the action of aldosterone on ENaC have primarily used transcriptional profiling of cultured cells. Here, we present a method to rapidly isolate collecting duct cells from the mouse kidney for transcriptional profiling. Our objective is to use this method, in combination with dietary sodium manipulation, to identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone.An antibody against the cell adhesion molecule L1‐CAM was previously used for fluorescence activated cell sorting (FACS) of collecting duct cells from human kidney. We verified L1‐CAM is also expressed in the collecting ducts of mouse kidney using immunofluorescence. We next developed a protocol for physical and enzymatic separation of mouse kidney into a single cell suspension. The kidney cells were labeled with L1‐CAM magnetic microbeads as well as antibodies against markers for endothelial cells (CD31) and immune cells (CD45). This allowed isolation of an L1‐CAM+ cell population using magnetic activated cell sorting (MACS), followed by separation of collecting duct cells from L1‐CAM+ endothelial and immune cells using FACS. RNA was purified from the FACS‐isolated collecting duct cells and, following poly(A) selection, used to generate Illumina TruSeq libraries. The libraries were sequenced on the Illumina HiSeq platform and the reads were aligned to the mouse genome using the Subread aligner. The aligned reads were mapped to transcripts using feature Counts and differentially expressed transcripts are identified using EdgeR.To identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone, C57BL/6J mice were fed sodium‐deficient diet (to stimulate endogenous aldosterone) or high‐sodium diet (to suppress endogenous aldosterone). After 5 days of dietary sodium manipulation, the kidneys were harvested, collecting duct cells were isolated, and RNA was sequenced and analyzed according to the above protocol. Of the differentially expressed transcripts, 234 were up‐regulated by aldosterone (more highly expressed in the sodium‐deficient diet samples) while 276 were down‐regulated by aldosterone (more highly expressed in the high‐sodium diet samples); false discovery rate < 0.05, no log2FC or log2CPM threshold. The transcriptome data were then compared with recently published single‐cell RNA‐seq data for the major collecting duct cell types, specifically intercalated cells and principal cells. It was found that 16 of the up‐regulated transcripts and 32 of the down‐regulated transcripts were highly expressed in principal cells, thereby limiting the number of candidate transcripts for subsequent validation. Of the transcripts that were highly expressed in principal cells, Sgk1 was the most differentially expressed transcript induced by aldosterone.Support or Funding InformationU.S. Department of Veterans Affairs: I01BX002228This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.