Abstract: PO1395
Single-Cell RNA Sequencing Reveals Transcriptomes of DCT1, DCT2, Macula Densa, and Two Subtypes of Cortical Thick Ascending Limb Cells
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolyte, and Acid-Base Disorders
- 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Chen, Lihe, National Heart Lung and Blood Institute, Bethesda, Maryland, United States
- Chou, Chung-Lin, National Heart Lung and Blood Institute, Bethesda, Maryland, United States
- Knepper, Mark A., National Heart Lung and Blood Institute, Bethesda, Maryland, United States
Background
Several distinct epithelial cell types have been proposed to form the transition region from the cortical thick ascending limb of Henle (CTAL) to the distal convoluted tubule (DCT) to the connecting tubule (CNT). However, a complete understanding of the cellular composition and transcriptional profiles of the cells in this region is lacking.
Methods
We developed a FACS protocol to enrich cells from the mouse CTAL-DCT-CNT region and carried out single-cell RNA-seq analysis (scRNA-seq) of 9099 such cells. We also used small-sample RNA-Seq to determine transcriptomes of microdissected tubules corresponding to 14 distinct mouse renal tubule segments.
Results
Unbiased clustering and UMAP visualization revealed a single cluster of cells showing Slc12a3 expression without Pvalb, which we identified as DCT2 cells. These cells express ENaC subunits but little or no Hsd11b2 or Aqp2 mRNA. These DCT2 cells also express Calb1, Slc8a1, S100g, Ptges, and Trpv5. In contrast, there were 6 tightly arranged clusters of cells expressing both Slc12a3 and Pvalb, which we identify as DCT1 cells. DCT1 heterogeneity appears to be associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. An additional DCT1 (Slc12a3+Pvalb+) cluster showed marked enrichment of cell cycle and cell proliferation associated mRNAs (e.g. Pcna, Mki67, Cdk1, and Top2a), which fits with the known plasticity of DCT cells. In addition, scRNA-seq identified three distinct CTAL (Slc12a1+) cell subtypes. One of these expressed Nos1, Avpr1a, and Pappa2, 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 types were also distinguished by alternative expression of Iroquois homeobox transcription factors, with Irx1 and Irx2 in the Cldn10+ CTAL cells and Irx3 in the Cldn16+ CTAL cells.
Conclusion
This work identifies unexpected diversity among cell types populating the CTAL and DCT. The new data have allowed the creation of a publicly accessible web resource for the support of future studies.
Funding
- Other NIH Support