Enriched Single-Nucleus RNA-Sequencing reveals unique attributes of distal convoluted tubule cells

Background The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. Methods To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining “Isolation of Nuclei TAgged in specific Cell Types” and NCC (sodium chloride cotransporter)-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting, and performed single nucleus transcriptomics. Results Among 25,183 DCT cells, 70% were from DCT1 and 30% from DCT2. Additionally, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a, Cenpp, and Mki67. Both DCT1 and DCT2 express NCC, magnesium transport genes are more abundant along DCT1; whereas calcium, electrogenic sodium and potassium transport genes are more abundant along DCT2. The transition between these two segments are gradual with a transitional zone where DCT1 and DCT2 cells are interspersed. The expression of the homeobox genes is not consistent between all DCT cells, suggesting that they develop along different trajectories. Conclusion Transcriptomics analysis of an enriched rare cell population using genetically targeted approach offers better clarification of the function and classification. The DCT segment is short, yet, can be separated into two sub-types that serve distinct functions, and are speculated to derive from different origins during development. Significance Statement High-resolution snRNAseq data indicate a clear separation between primary sites of calcium and magnesium handling within DCT. Both DCT1 and DCT2 express Slc12a3 , but these subsegments serve distinctive functions, with more abundant magnesium handling genes along DCT1 and more calcium handling genes along DCT2. The data also provides insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand.