Spatial characterization of sex differential regulations in kidney across lifespan

There is a clear sex bias in the incident rates and progressions of many kidney diseases and kidney cancers. To obtain a comprehensive understanding of the genetic and epigenetic regulation of kidney sexual dimorphism, we integrated single nucleus RNA and ATAC sequencing (snRNA-Seq and snATAC-Seq) and Visium spatial transcriptomics (ST) data to build a spatially resolved cell-type-specific molecular atlas for the mouse kidney throughout the lifespan, from embryo, newborn, youth, adolescence, adult to old age, of both sexes. We demonstrate that proximal tubules (PT) have the most sex-biased differentially expressed genes (DEGs) (164 and 227 for male and female respectively), which appear after 3 weeks of age and are associated with hormonal regulations. Notably, we reveal the potential mechanism of indirect and direct involvement of estrogen and androgen, respectively, in sex-biased gene expression regulations in the kidney. Specifically, regulon analysis shows that a set of female-biased DEGs, including Socs2, might be indirectly regulated by estrogen through prolactin-induced Jak2/Stat5 activation. On the other hand, chromatin accessibility investigation identifies androgen receptor motifs as significantly enriched in males, might be directly involved in regulating male-biased DEGs, such as Slco3a1, Serpinf2, Slc7a13, Slc22a30, Cyp4b1, and Acsm2. Sex-biased DEGs identified from mice, including several not previously highlighted, were validated by immunofluorescence and multiplex imaging data in human kidneys. Moreover, older male mice (92 weeks) display more aging-related gene alterations in loops of Henle and PTs, while older females have more aging-related gene alterations in fibroblasts. Our results provide the community with rich resources and better understanding of spatially resolved gene expression and hormone regulation for sexual dimorphism in the kidney across the lifespan. C57BL/6J mice were originally purchased from the Jackson Laboratory. Mouse kidneys were collected at embryos (E16.5), newborns (P0), 3 weeks (W3), 12 weeks (W12), 52 weeks (W52), and 92 weeks (W92). To get E16.5 kidneys, 4 female mice and 1 male mouse were grouped for mating on day 1 at 5 pm. Then the male was removed from the females on day 2 at 10 am. Day 2 was designated as E0.5 for any pregnant females. For kidneys collected from other ages, mice were purchased from the Jackson Laboratory. Mice at E16.5 and P0 were euthanized by decapitation. Older mice were euthanized by carbon dioxide asphyxiation. For each mouse, one kidney was embedded in optimal cutting temperature compound (OCT) and the other one was fixed in 10% neutral buffered formalin (Epredia, 5725) then embedded in paraffin. For Visium ST, 10 μm mid-sagittal OCT sections were used on the 10x Genomics Visium platform; for snRNA-Seq and snATAC-Seq, pooled snap-frozen tissues were used for E16.5 and P0 mice, and 300 μm of mid-sagittal OCT sections were used for mice from W3 to W92. All animal experiments were approved by the Washington University in Saint Louis Institutional Animal Care and Use Committee (IACUC) office.