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

Abstract

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.

Keywords: distal convoluted tubule; macula densa; scRNA-seq; thick ascending limb.

Graphical abstract

Figure 1.

scRNA-seq of DCT cells. (A) Overview of protocol for the enrichment of DCT cells for single-cell analysis. (B) Immunostaining showing the distribution of Slc12a3 (red) and Pvalb (green) in microdissected CTAL to CNT region. Nuclei are stained using DAPI (blue). Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Distribution pattern of Embigin transcript along the mouse renal nephron segments. The tubules in the renal cortex are shown in shaded yellow. TPM, Transcripts Per Million. (D) Embigin was used as a positive surface marker for DCT and thick ascending limb cells and a negative marker for proximal tubule cells and principal cells. (E) UMAP projection of cells from all three 10× Chromium datasets. Different clusters are colored and labeled. Four major clusters are highlighted by dashed circles and annotated on the basis of (F). (F) Violin plots of renal cell marker genes across all clusters. Cluster information is shown at the top.

Figure 2.

Heterogeneity of DCT cells revealed by scRNA-seq. (A) Dot plot showing the distribution of Slc12a3, Pvalb, and Aqp2 expression across the clusters. Data are normalized and scaled (z score) to examine relative expression across the cell clusters; “avg.exp” is the z score of the average gene expression of all cells within a cluster (scaled values), and “pct.exp” is the percentage of cells with nonzero gene expression. (B) UMAP projection for Slc12a3 and Pvalb expression. Arrows point to DCT2 cells; arrowheads point to CNT cells. (C) Heat map showing the top five genes in DCT and CNT cell clusters. Data are scaled (z score). A similar analysis is also provided in Supplemental Figure 2.

Figure 3.

Distributions of transcripts associated with major transport pathways in DCT cells. (A) UMAP projection for Stmn1, Mki67, and Lrrc52. (B) Distribution of major transport pathways in DCT cells; “avg.exp” is the z score of the average gene expression of all cells within a cluster (scaled values), and “pct.exp” is the percentage of cells with nonzero gene expression. (C) Immunostaining showing Scnn1b and Scnn1g expression in microdissected tubule from the CTAL-DCT-CNT region. Scnn1b and Scnn1g are in green, Slc12a3 is in purple, Pvalb is in cyan, and DNA is in blue. Arrows point to regions at the point of initial expression of Scnn1b and Scnn1g. Scale bars, 50 µm. (D) UMAP projection for Emx1, Sall3, and Hoxb7.

Figure 4.

Single-cell analysis of CTAL cells. (A) UMAP visualization of Slc12a1. (B) UMAP plot for Nos1, Avpr1a, and Pappa2. (C) Heat map showing the top 20 marker genes in C5, C6, and C14. Each column indicates a cell. Colors indicate the expression level (z score). (D) Immunostaining showing Slc12a1 (green) and Cldn10 (purple) on a microdissected mouse TAL. Nuclei are stained with DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole. The feature plot for Cldn10 is shown in the right panel. Scale bars, 10 µm. (E) Immunostaining coupled with in situ hybridization (RNAscope) showing Slc12a1 (white), Ptger3 (green), and Foxq1 (red) on mouse kidney section. Nuclei are stained with DAPI (blue). Arrows point to Foxq1⁺-positive cells (red). Lower-magnification views are provided in Supplemental Figure 4 and can be viewed at https://esbl.nhlbi.nih.gov/MRECA/Supplement/. Scale bars, 20 µm. (F) UMAP visualization of Irx1, Irx2, Irx3, Kcnj10, and Jag1.