Targeted Single-Cell RNA-seq Identifies Minority Cell Types of Kidney Distal Nephron
Lihe Chen, Chun-Lin Chou, Mark A. Knepper
Abstract
Significance Statement A major objective in modern biology is generation of comprehensive atlases of various organs that identify all cell types and their expressed genes. In the kidney, extensive data describe proximal tubule and collecting duct cells but not the rarer intermediate epithelial cell types. Coupling of a cell enrichment protocol with single-cell RNA-seq analysis resolved the cellular composition and transcriptional profiles of the minority epithelial cell types of mouse kidney distal nephron. These data are provided in user-friendly websites that enable the mapping and comparison of genes of interest among cell types and renal tubule epithelia. Background Proximal tubule cells dominate the kidney parenchyma numerically, although less abundant cell types of the distal nephron have disproportionate roles in water and electrolyte balance. Methods Coupling of a FACS-based enrichment protocol with single-cell RNA-seq profiled the transcriptomes of 9099 cells from the thick ascending limb (CTAL)/distal convoluted tubule (DCT) region of the mouse nephron. Results Unsupervised clustering revealed Slc12a3 + / Pvalb + and Slc12a3 + / Pvalb − cells, identified as DCT1 and DCT2 cells, respectively. DCT1 cells appear to be heterogeneous, with orthogonally variable expression of Slc8a1 , Calb1 , and Ckb . An additional DCT1 subcluster showed marked enrichment of cell cycle–/cell proliferation–associated mRNAs ( e.g ., Mki67 , Stmn1 , and Top2a ), which fit with the known plasticity of DCT cells. No DCT2-specific transcripts were found. DCT2 cells contrast with DCT1 cells by expression of epithelial sodium channel β - and γ -subunits and much stronger expression of transcripts associated with calcium transport ( Trpv5 , Calb1 , S100g , and Slc8a1 ). Additionally, scRNA-seq identified three distinct CTAL ( Slc12a1 + ) cell subtypes. One of these expressed Nos1 and Avpr1a , consistent with macula densa cells. The other two CTAL clusters were distinguished by Cldn10 and Ptger3 in one and Cldn16 and Foxq1 in the other. These two CTAL cell types were also distinguished by expression of alternative Iroquois homeobox transcription factors, with Irx1 and Irx2 in the Cldn10 + CTAL cells and Irx3 in the Cldn16 + CTAL cells. Conclusions Single-cell transcriptomics revealed unexpected diversity among the cells of the distal nephron in mouse. Web-based data resources are provided for the single-cell data.