Transcriptomes of major renal collecting-duct cell types in mouse identified by single-cell RNA-Seq

Abstract

Prior RNA sequencing (RNA-Seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume and extracellular fluid composition. To enrich these cell types, we used fluorescence-activated cell sorting (FACS) that employed well established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, viz. c-Kit. Single-cell RNA-Seq using the 1C- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs and PCs. The data were used to create a freely-accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type including cell-surface receptors, transcription factors, transporters and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing both aquapor¡n-2 and either anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g. Notch2 chiefly in PCs vs Jag1 chiefly in ICs) suggesting signaling crosstalk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.SIGNIFICANCE STATEMENTA long-term goal in mammalian biology is to identify the genes expressed in every cell type of the body. In kidney, the expressed genes (“transcriptome”) of all epithelial cell types have already been identified with the exception of the cells that make up the renal collecting duct, responsible for regulation of blood pressure and body fluid composition. Here, a technique called "single-cell RNA-Seq" was used in mouse to identify transcriptomes for the major collecting-duct cell types: type A intercalated cells, type B intercalated cells and principal cells. The information was used to create a publicly-accessible online resource. The data allowed identification of genes that are selectively expressed in each cell type, informative for cell-level understanding of physiology and pathophysiology.

Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption

“…Coupled with our expression studies (Results) we would like to propose that Slc4a8 is not expressed in the CCD, including the B-intercalated cells. Further support for the absence of Slc4a8 in the cortical collecting duct was reported in a recent study, which indicated that in single cell isolation of CCD cells (A-IC, B-IC and principal cells) Slc4a8 was not detected [31]. Given the complete absence of Slc4a8 mRNA in the CCD [31], it is highly likely that the signal detected in western blots using .…”

“…Further support for the absence of Slc4a8 in the cortical collecting duct was reported in a recent study, which indicated that in single cell isolation of CCD cells (A-IC, B-IC and principal cells) Slc4a8 was not detected [31]. Given the complete absence of Slc4a8 mRNA in the CCD [31], it is highly likely that the signal detected in western blots using . From a thermodynamic point of view, the parallel activation of pendrin and NHE-2 is equivalent to the coupling of an NBC-like pathway with pendrin, in that both pathways lead to enhanced entry of sodium and chloride in an electroneutral manner.…”

Understanding the Biology and Pathogenesis of the Kidney by Single-Cell Transcriptomic Analysis

“…To overcome these potential problems, it is feasible to dissect only a specific portion of kidney tissues (e.g., proximal tubules or glomeruli) and to dissociate cells with an improved method for increased cell viability, such that the complexity of cell types will be reduced and cell viability will be increased, allowing the rare cell types to be picked up and analyzed. Indeed, Chen et al [76] focused on only collecting duct for scRNA-seq and resolved the A-type intercalated cells, the B-type intercalated cells, principal cells, and hybrid cells that are between A-and B-type intercalated cells in gene expression. This approach gave rise to more precise dissection of cell types or subtypes compared with scRNA-seq of whole kidney, as described in the work by Park et al [79].…”

“…Chen et al [76] performed scRNA-seq on mouse renal collecting duct cells and created the database from which genes selectively expressed in each cell type of collecting duct can be found. Furthermore, they showed that a small fraction of hybrid cells expressed aquaporin-2 and anion exchanger 1 or pendrin transcripts.…”

Novel Proinflammatory Function of Renal Intercalated Cells

“…ICs were isolated by fluorescence activated cell sorting from previously characterized transgenic mice that express enhanced green fluorescent protein (EGFP) specifically in these cells (B1-EGFP mice) [35]; no expression was detected in all other renal cell types [1]. In addition, RNA sequencing of isolated EGFP ICs identified P2Y 14 as one of the most expressed genes in these cells, while single cell RNA sequencing showed specific expression of P2Y 14 in type A ICs [36]. Subsequent examination showed that P2Y 14 is located on the apical surface of ICs in contact with the tubular fluid (pre-urine; Fig.…”