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

Abstract

BackgroundHuman nephrogenesis is complete at 34-36 weeks gestation, with 60% of nephrons forming during the third trimester through lateral branch nephrogenesis (LBN). Currently, no mechanism exists for LBN as there are no late gestation human kidney transcriptional datasets. We hypothesized that a differentiated but dividing population of nephron progenitor cells (NPCs) would contribute to the amplification of nephrons in late gestation. We used the rhesus macaque, an established model of LBN, to help identify potential mechanisms. MethodsSingle-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. ResultsscRNA-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 ureteric bud (UB) tip, suggesting a compositional shift in WNT and SEMA signaling components within the naive NPC population during LBN. ConclusionThe rhesus macaque uniquely enables molecular studies of late-gestation primate nephrogenesis. Our study suggests the hypothesis that a transitional state of self-renewing NPCs 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. Translational statementNo transcriptional data exists for the late-gestation human kidney, during which 60% of the nephron endowment is formed through a primate-specific process called lateral branch nephrogenesis (LBN). In this study, we used single-cell RNA sequencing the late gestation rhesus macaque as a model for this developmental stage. Our findings suggest a potential mechanism for LBN, in which a transitional state of self-renewing nephron progenitor cells (NPCs) is supported by compositional shifts in key pathways, allowing for continued nephron formation in late gestation.