Defining Stromal Cell Heterogeneity in the Kidney with Single-Nucleus RNA Sequencing

Background: The kidney stroma are critical perivascular cells that support multiple kidney functions such as regulation of regional blood flow and maintenance of the glomerular filtration barrier. While they are commonly identified by abundant expression of Pdgfrb, recent studies have hinted at more heterogeneity. We examined their heterogeneity by performing single-nucleus RNA sequencing (snRNAseq) on nuclei that were enriched for kidney stromal cells with the goal of defining robust gene markers for unique stromal cell subpopulations. Methods: Pdgfrb-creERT2 mice were bred to the INTACT mouse to create Pdgfrb-INTACT mice which express GFP attached to a nuclear envelope protein within stromal cells. We isolated nuclei from Pdgfrb-INTACT mice and enriched for GFP expressing nuclei with Fluorescent Activated Nuclei Sorting and then transcriptionally profiled them on the 10X Genomics platform. The resulting transcriptome data was analyzed with Seurat to perform dimensional reduction which delineated unique populations of stromal cells. We then identified differentially expressed genes (DEGs) that defined each subpopulation. The specificity of these DEGs was confirmed by RNAscope. We then analyzed a separate kidney snRNAseq dataset that was not enriched for Pdgfrb nuclei to determine whether these unique stromal cell populations were present. Results: We transcriptionally profiled 59,349 nuclei from Pdgfrb-INTACT mice which divided into 11 subpopulations, including 4 populations of fibroblasts, 4 populations of contractile cells, 2 populations of mesangial cells, and a proliferating population. Kcnd3, 6530403H02Rik, Itgbl1, Ahrr delineated the fibroblast populations; Hpse2, Tenm2, Lhfpl3, and Mannr delineated the contractile populations; and Grip1 and Limch1 delineated the mesangial subpopulations. We localized these distinct stromal cell subpopulations in mouse kidney using population-specific DEGs with RNAscope. Analysis of a separate kidney snRNAseq dataset revealed subpopulations with similar gene expression profiles to the subpopulations we identified in the Pdgfrb-INTACT mice. Conclusion: Stromal cells are functionally and genetically diverse and can be characterized through distinct genetic markers. Enriching for stromal cells with the Pdgfrb-INTACT mouse enabled us to identify robust genetic markers of the kidney stroma that have been previously unrecognized. Funding: NIDDK Support, Private Foundation Support