Uncovering Distal Convoluted Tubule (DCT) Heterogeneity with Enriched DCT Single‐Nucleus RNA‐Sequencing
Authors: Su Xiao‐Tong , McCormick James A. , Yang Chao‐Ling , Ellison David H. , Nelson Jonathan 
Published: May 2022

The mammalian kidneys play a critical role in regulation of electrolyte balance and blood pressure. At least 14 kidney tubule segments are described, of which the distal convoluted tubule (DCT) is the shortest; yet this short segment plays a crucial role in a variety of homeostatic processes, including sodium, potassium, calcium and magnesium handling. The DCT is morphologically and functionally heterogeneous and comprises DCT1 and DCT2. A “shotgun” single‐cell RNA sequencing (scRNAseq) approach transcriptionally profiles all cells from a piece of kidney tissue, which may allow abundant cell types to overwhelm minority cell types, such as DCT, thereby masking their complex heterogeneity. Recently, Chen et al. used the targeted scRNAseq analysis and identified 6 populations of DCT1 cells; yet the DCT2 was not well represented and therefore poorly resolved.

To understand the heterogeneity within the DCT2 population, we used “Isolation of Nuclei TAgged in specific Cell Types” (INTACT), which fluorescently labels nuclei from genetically targetable cells. Combined with the NCC (sodium chloride cotransporter)‐driven inducible Cre recombinase, DCT nuclei can be enriched using Fluorescence‐Activated Nuclei Sorting (FANS). Female NCC‐Cre‐INTACT mice were treated with Tamoxifen to induce expression of the GFP‐tagged SUN1, a nuclear membrane protein. Mice were provided either normal (NK) or potassium deficient diet (KD) for 4 days and kidneys were harvested. Nuclei from one kidney cortex were isolated and 10,000 GFP‐positive nuclei from each mouse were input into 10X Genomics Chromium Controller and the libraries were sequenced using NovaSeq. The resulting dataset was analyzed using Seurat to determine the number of populations and their identities using the UMAP algorithm for analysis and visualization.

After data quality control, we detected 24,054 genes across 70,233 cells. Unbiased clustering and UMAP visualization revealed 12 clusters, with most cells from the DCT (78%) indicating the success of the enrichment process. Among those, 70% of the cells were from DCT1 and 30% from DCT2. There was clear separation of DCT1 from DCT2, with DCT1 enriched in Trpm7, Egf, Umod and Erbb4, and DCT2 in Slc8a1, Calb1, S100g, Trpv5, and Scnn1g. After further analysis of the DCT2 population, we identified two specialized populations, DCT2‐α and DCT2‐β. The DCT2‐α is enriched in NCC, magnesium and calcium‐related genes (Slc12a3, Wnk1, Slc8a1, Calb1, Trpm6, etc.) whereas DCT2‐β is enriched in mineralocorticoid receptors and epithelial sodium channel‐related genes (Nr3c2, Klk1, Scnn1b, Scnn1g, etc.), indicating that DCT2‐α is more similar to DCT1 and DCT2‐β to collecting duct. Four‐day KD treatment decreased the Klk1, Scnn1b and Scnn1g expression levels in DCT2‐β, indicating that some DCT2 cells may reprogram to retain potassium in response to the short‐term potassium deficiency.

Our results suggest that DCT2 cells fall into specialized populations that likely serve different functions in electrolyte handling. The DCT2 is plastic and can transform from potassium excretion to potassium sparing in response to dietary potassium restriction to maintain electrolyte balance.