Targeted Single-Cell RNA-seq Identifies Minority Cell Types of Kidney Distal Nephron.

Abstract

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. 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. Unsupervised clustering revealed <i>Slc12a3</i> ⁺/<i>Pvalb</i> ⁺ and <i>Slc12a3</i> ⁺/<i>Pvalb</i> ^- cells, identified as DCT1 and DCT2 cells, respectively. DCT1 cells appear to be heterogeneous, with orthogonally variable expression of <i>Slc8a1</i>, <i>Calb1</i>, and <i>Ckb</i>. An additional DCT1 subcluster showed marked enrichment of cell cycle-/cell proliferation-associated mRNAs (<i>e.g</i>., <i>Mki67</i>, <i>Stmn1</i>, and <i>Top2a</i>), 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 <i>β</i>- and <i>γ</i>-subunits and much stronger expression of transcripts associated with calcium transport (<i>Trpv5</i>, <i>Calb1</i>, <i>S100g</i>, and <i>Slc8a1</i>). Additionally, scRNA-seq identified three distinct CTAL (<i>Slc12a1</i> ⁺) cell subtypes. One of these expressed <i>Nos1</i> and <i>Avpr1a</i>, consistent with macula densa cells. The other two CTAL clusters were distinguished by <i>Cldn10</i> and <i>Ptger3</i> in one and <i>Cldn16</i> and <i>Foxq1</i> in the other. These two CTAL cell types were also distinguished by expression of alternative Iroquois homeobox transcription factors, with <i>Irx1</i> and <i>Irx2</i> in the <i>Cldn10</i> ⁺ CTAL cells and <i>Irx3</i> in the <i>Cldn16</i> ⁺ CTAL cells. 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.