MULTIOMIC ANALYSIS OF HUMAN KIDNEY DEVELOPMENT TO IDENTIFY DRIVERS OF CELL FATE

Congenital Anomalies of the Kidney and the Urinary Tract (CAKUT) are a diverse group of structural urinary tract malformations that affect 1 in 500 live births. CAKUT are the leading cause of kidney failure in children and young people. While over 50 monogenic causes have been identified, many of which encode transcription factors (TFs), >80% of cases remain unexplained. We hypothesise that disruptions in cell-type and developmental stage specific gene-regulatory networks are important in the pathogenesis of CAKUT. We aimed to identify key driver TFs in human embryos at critical timepoints for normal kidney development to prioritise candidate CAKUT genes.

We performed paired single-nucleus RNA-seq and ATAC-seq (10X Multiome) on normal human embryonic kidneys and urinary tracts at 6 and 8 post-conception weeks (6PCW, n=1; 8PCW, n=3) obtained from the MRC-Wellcome Trust Human Developmental Biology Resource. Seurat and Signac were used for pre-processing, quality control, normalisation, dimensionality reduction and clustering. Automated annotation was performed with Azimuth and refined manually using published cell markers. Monocle3 was used to perform pseudotime trajectory inference and differential expression analysis. The differentially expressed genes were filtered against the DoRothEA human TF database to identify lineage-specific driver TFs. Rare variant association testing was performed using SAIGE-GENE+ using Genomics England whole-genome sequencing data (110 cases with cystic dysplasia and 25,302 controls).

7,717 (6PCW) and 14,222 (8PCW) high-quality nuclei were obtained. Clustering and annotation identified key cell populations, including metanephric mesenchyme and ureteric bud (Figure 1). Pseudotime trajectory analysis indicated that the intermediate mesoderm bifurcates into the gonadal ridge and mesenchymal progenitor lineages at 6PCW. By 8PCW, trajectory reconstruction revealed branching of the ureteric bud into two urothelial lineages and differentiation of PAX8+/PAX8- metanephric mesenchyme through an early tubular state into podocytes. Integrated single-nucleus transcriptome and chromatin accessibility analysis identified NR2F2 and GRHL2 as potential candidate driver TFs.

NR2F2 was highlighted along the PAX8+/PAX8- metanephric mesenchyme to podocyte differentiation trajectory. GRHL2 was also seen in this trajectory as well as the ureteric bud to urothelial lineage. Integrated analysis showed widespread NR2F2 expression at 6PCW, with strong enrichment in the PAX8+/PAX8- metanephric mesenchyme by 8PCW. GRHL2 showed high expression and accessible chromatin in the intermediate mesoderm at 6PCW, and in early tubular, ureteric bud, and urothelial populations by 8PCW. In Genomics England data, NR2F2 is associated with cystic dysplasia (p=0.0054) and loss-of-function variants in this gene are reported to cause congenital anomaly syndromes (PMID: 37500725). GRHL2 also demonstrated nominal association with unsolved cystic kidney disease (p=0.04) and its deletion in mice worsens cyst growth (PMID: 38656794). 

We show how multiomic analysis of early kidney development can be used to identify potential drivers of cell fate and prioritise candidate CAKUT genes. Characterising the gene-regulatory mechanisms important for normal kidney development is vital to better understand what might go wrong in patients with CAKUT.