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 m 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 m 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.多种肾脏疾病及肾癌的发病率与病情进展存在显著的性别偏向性。为全面解析肾脏性别二态性的遗传与表观遗传调控机制，本研究整合了单细胞核RNA测序与转座酶可及性测序（single nucleus RNA and ATAC sequencing，snRNA-Seq和snATAC-Seq）以及Visium空间转录组（spatial transcriptomics，ST）数据，构建了雌雄小鼠整个生命周期（胚胎期、新生期、青年期、青春期、成年期至老年期）内的空间分辨细胞类型特异性分子图谱。研究发现，近端小管（proximal tubules，PT）的性别偏向性差异表达基因（differentially expressed genes，DEGs）数量最多（雄性与雌性分别为164个和227个），此类基因于3周龄后开始表达，并与激素调控密切相关。值得注意的是，本研究揭示了雌激素与雄激素分别通过间接与直接途径参与肾脏性别偏向性基因表达调控的潜在机制：调控元分析（regulon analysis）显示，包括Socs2在内的一批雌性偏向性DEGs可能通过催乳素诱导的Jak2/Stat5激活通路，间接受雌激素调控；而染色质可及性（chromatin accessibility）分析发现，雄激素受体基序在雄性样本中显著富集，可直接参与调控雄性偏向性DEGs，如Slco3a1、Serpinf2、Slc7a13、Slc22a30、Cyp4b1及Acsm2。本研究在小鼠中鉴定出的性别偏向性DEGs（含此前未被报道的多个基因）已通过人类肾脏组织的免疫荧光（immunofluorescence）与多重成像（multiplex imaging）数据得到验证。此外，92周龄的老年雄性小鼠在亨利袢与近端小管中表现出更多衰老相关的基因改变，而老年雌性小鼠则在成纤维细胞中出现更多此类变化。本研究结果为科研共同体提供了丰富的资源，有助于更深入理解生命周期内肾脏性别二态性的空间分辨基因表达与激素调控机制。C57BL/6J小鼠（C57BL/6J mice）最初购自杰克逊实验室（Jackson Laboratory）。实验分别于胚胎期（E16.5）、新生期（P0）、3周龄（W3）、12周龄（W12）、52周龄（W52）及92周龄（W92）收集小鼠肾脏。为获取E16.5胎鼠肾脏，于第1日下午5时将4只雌性小鼠与1只雄性小鼠合笼；第2日上午10时移除雄性小鼠，将该日定为受孕小鼠的E0.5时期。其余年龄组小鼠均直接从杰克逊实验室购得。E16.5与P0阶段的小鼠通过断头法实施安乐死，老年小鼠则采用二氧化碳窒息法安乐死。每只小鼠取一侧肾脏用最优切割温度包埋剂（optimal cutting temperature compound，OCT）包埋，另一侧肾脏用10%中性缓冲福尔马林（10% neutral buffered formalin，Epredia, 5725）固定后进行石蜡包埋。Visium空间转录组实验采用10 μm厚度的正中矢状位OCT切片，在10x Genomics Visium平台上完成；单细胞核RNA测序与转座酶可及性测序实验中，E16.5与P0阶段小鼠采用混合速冻组织样本，W3至W92阶段小鼠则采用300 μm厚度的正中矢状位OCT切片。所有动物实验均经圣路易斯华盛顿大学实验动物护理与使用委员会（Washington University in Saint Louis Institutional Animal Care and Use Committee，IACUC）批准。