Bridging the gap of late-gestation nephrogenesis using a non-human primate model

Background: Prematurity is associated with low nephron endowment and an increased risk of chronic kidney disease. Human nephrogenesis is complete at 34-36 weeks gestation, with 60% of nephrons forming during the third trimester through lateral branch nephrogenesis (LBN). We hypothesized that a differentiated but dividing population of nephron progenitor cells (NPCs) would contribute to the amplification of nephrons in late gestation. Methods: Single-cell RNA-sequencing (scRNA-Seq) was performed on cortically-enriched fetal rhesus kidneys (n=9) from late second trimester and third trimester during LBN. This data was integrated with publicly available human scRNA-seq datasets from 8-18 weeks gestation kidneys (n=8) using state-of-the-art bioinformatics pipelines. Differentially expressed genes and ligand-receptor interactions were assessed and validated using RNAScopeTM on human and rhesus archival tissue. Results: scRNA-Seq of 64,782 rhesus cells revealed 37 transcriptionally distinct cell populations, including 7,879 rhesus NPCs. Pseudotime analyses identified a late gestation-specific lineage branch of differentiated NPC in rhesus that was not observed in mid-gestation humans. Differential expression analyses identified increased SFRP1, FZD4, and TLE2 and decreased FZD7, SHISA2, SHISA3, and TLE4 within the late-gestation rhesus NPC compared to mid-gestation human NPC and increased SEMA3D within the rhesus UB tip, suggesting a compositional shift in WNT and SEMA signaling components within the naive NPC population during LBN. Conclusion: The rhesus macaque uniquely enables molecular studies of late-gestation primate nephrogenesis. Our study suggests the hypothesis that a transitional state of self-renewing NPC supported by compositional shifts in key pathways may underlie the switch from branching phase nephrogenesis to lateral branch nephrogenesis and support ongoing nephron formation in late gestation.