Sex-Dependent Dynamic Transcriptional Responses in AKI in Mice

Background: Previous studies demonstrated the role of sex differences in protection from acute kidney injury (AKI). However, the mechanism(s) that increase the susceptibility to AKI in males vs females remains unclear. We aim to investigate the sex-specific transcriptomic differences with single nucleus multiome sequencing data to identify mediators that increases the susceptibility to AKI in males vs females post-aristolochic acid I (AAI) injury in mice. Methods: Male and female mice were injected with one dose of DMSO or AAI (2mg/kg) and perfused after 72 hours. Using 10X Genomics, single nuclear multiome (scRNA and scATAC) sequencing on the kidney samples was performed. CellRanger-ARC aligned the sequencing data and proceeded through the ArchR pipeline for data mining. Alevin-fry generated splice counts on the original data and scVelo performed RNA velocity analysis. Kidneys were stained for FOSL1 and JUNB by immunofluorescence. Results: Compare to females, 4 unique PT clusters formed in males after AAI. Two of these were similar to uninjured PT clusters, but the other two showed increased expression of injury markers, including AP-1 transcription factors(TF) (Fosl1, Junb; injury cluster), NF-kB signaling pathway (Rela, Relb, Nfkb1, Nfkb2; intermediate cluster). Immunostaining validated expression of these unique proteins in injured PT clusters in male mice vs female mice post-AAI. The injury cluster demonstrated high expression of Junb, as well as motif enrichment and footprinting bias. Consistent with its role as an early response gene, RNA velocity showed that Junb was already fully spliced, suggesting earlier upregulation. By contrast Fosl1, had lower expression, but a higher RNA velocity, suggesting ongoing upregulation. Fosl1 motifs were already open, suggesting that chromatin in these cells was already primed for Fosl1 binding. In the intermediate cluster, NFKB pathway genes showed less well defined dynamics, but enrichment for open motifs and footprinting analysis suggested active transcription. Analysis of downstream target genes showed high expression and dynamics of Plau, Tnfaip3, and Vcam1. Expression, motifs, and footprinting analyses for these TFs in female PT clusters after AAI were all negative. Conclusion: AKI induced dynamic AP-1 and NFKB transcriptional activity in two distinct injured PT clusters in male, but not female mice. Funding: NIDDK Support, Veterans Affairs Support