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 RNAScope™ 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.

Key points

• Single-cell RNA sequencing analysis of late-gestation primate nephron progenitor cells (NPCs) identifies rhesus-specific self-renewing NPCs that are not observed in mid-gestation humans.

• Differential expression and ligand-receptor interactions suggest a compositional shift in WNT signaling within the naive NPC population during LBN.

• FZD7 localized to the connecting segment and SEMA3D was identified in the ureteric bud (UB) tips of late-gestation primates.