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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 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, c-Kit. Single-cell RNA-seq using the IC- 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 aquaporin-2 and 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 cross-talk 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.

SUBMITTER: Chen L

PROVIDER: S-EPMC5699061 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Chen Lihe L Lee Jae Wook JW Chou Chung-Lin CL Nair Anil V AV Battistone Maria A MA Păunescu Teodor G TG Merkulova Maria M Breton Sylvie S Verlander Jill W JW Wall Susan M SM Brown Dennis D Burg Maurice B MB Knepper Mark A MA

Proceedings of the National Academy of Sciences of the United States of America 20171031 46

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 FACS that empl ...[more]

PMID: 29089413

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Project description:Purpose: Transport processes in the renal collecting duct are responsible for precise regulation of blood pressure and body fluid composition. The collecting duct is composed of at least three cell types, type A intercalated cells (A-IC), type B intercalated cells (B-IC) and principal cells (PC). To identify the genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters and secreted proteins, we used cell surface markers necessary for isolation of each of the three cell types using fluorescence-activated cell sorting and carried out single-cell RNA-Seq to measure the mRNA species in each of these cell types. Methods: We enriched each of the three cell types using fluorescence-activated cell sorting. Subsequently, we carried out single-cell RNA-Seq of those cells using a microfluidic chip (Fluidigm C1 system). Single-cell cDNA libraries were constructed for paired-end sequencing and sequenced on Illumina HiSeq3000 platform. In addition, we also microdissected mouse CCDs and cTALs and carried out single-tubule RNA-Seq. Reads were mapped to mouse Ensembl Genome by STAR and transcript abundances were calculated in the units of transcripts per million (TPM) using RSEM (https://github.com/deweylab/RSEM). Single-cell RNA-Seq data anaysis were carried out using Seurat package in R. Results and conclusion: Single-cell cDNA libraries were constructed for paired-end sequencing and at least 10-million sequence reads per cell were mapped to the mouse genome (Ensembl, GRCm38.p5). On average, cells were sequenced to a depth of 3000 genes (TPM>1). Unsupervised clustering analysis revealed five different cell types, namely type A intercalated cells (n=87), type B intercalated cells (n=23), principal cells (74), proximal tubule cells (n=19), and non-epithelial cells (n=32). The identified patterns of gene expression among A-ICs, B-ICs and PCs provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

Dataset Information

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

Publications

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

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