Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin

The global burden of chronic kidney diseases is rapidly increasing and is projected to become the fifth most common cause of years of life lost worldwide by 2040. Sexual dimorphism in kidney diseases and transplantation is well known, yet sex-specific therapeutic strategies are still missing. One reason is the lack of knowledge due to the lack of inclusion of sex as a biological variable in study designs. This work aimed at identification of molecular signatures of male and female podocytes, gate-keepers of the glomerular filtration barrier. Like cardiomyocytes, podocytes are terminally differentiated cells which are highly susceptible towards pathological challenges. Podocytes are the decisive cell-type of the kidney to maintain the physiological blood-urine barrier, and disturbances of their homeostasis critically accelerate kidney function impairment. By help of a genomic mouse model, highly purified podocytes were obtained from male and female mice with and without pharmacological challenge of the mechanistic target of rapamycin (mTOR) signaling pathway which is known to be deregulated in major kidney diseases. Deep RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. Remarkably, high number of previously reported kidney disease genes showed so far unknown intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. Our work provides an in-depth database for novel targets to be tested in kidney disease models to advance with sex-specific treatment strategies. Overall design: In order to investigate cell type specific molecular response upon mTOR inhibition, podocytes were isolated from ROSAmT/mG-NPHS2 Cre mouse female and male mice treated with and without rapamycin followed by mRNAseq.

慢性肾脏病（chronic kidney diseases）的全球疾病负担正快速攀升，预计到2040年将成为全球范围内第五大导致寿命损失年（years of life lost）的病因。肾脏病与移植中的性别二态性（sexual dimorphism）已被广泛认知，但目前仍缺乏针对性别的治疗策略。究其原因之一，是现有研究设计未将性别作为生物学变量纳入，导致相关研究认知不足。本研究旨在鉴定雄性与雌性足细胞（podocytes）的分子特征——足细胞是肾小球滤过屏障（glomerular filtration barrier）的核心守门人。与心肌细胞类似，足细胞属于终末分化细胞（terminally differentiated cells），对病理应激具有高度易感性。足细胞是维持生理性血液-尿液屏障的关键细胞类型，其稳态失衡会显著加速肾功能损伤进程。本研究借助基因组学小鼠模型，从经/未接受哺乳动物雷帕霉素靶蛋白（mechanistic target of rapamycin, mTOR）信号通路药理学干预的雌雄小鼠体内，分离得到高纯度足细胞；而mTOR信号通路失调已被证实与多种重大肾脏病密切相关。通过深度RNA测序（deep RNA sequencing）、蛋白质组学（proteomics）与代谢组学（metabolomics）分析，研究团队发现线粒体、翻译相关及结构相关转录本的表达水平、蛋白质丰度，以及代谢通路调控均存在显著的内源性性别差异，这些差异可能从根本上参与肾脏病易感性与进展过程中的性别差异形成。值得注意的是，大量此前已报道的肾脏病相关基因，其内在性别二态性以及对mTOR抑制的响应模式此前均未被发现。本研究构建了一套深度数据库，可为肾脏病模型中新型治疗靶点的验证提供支撑，以推动针对性别特异性治疗策略的开发。整体实验设计：为探究mTOR抑制下的细胞类型特异性分子响应，本研究分别从经/未接受雷帕霉素（rapamycin）处理的ROSAmT/mG-NPHS2 Cre转基因雌雄小鼠体内分离足细胞，随后开展mRNA测序（mRNAseq）。