Role of nonmuscle myosin IIA in sex-dependent failed repair mechanisms in acute kidney injury

摘要 推动急性肾损伤(AKI)向慢性肾脏疾病(CKD)进展的细胞机制 都没有被很好地理解。Aki是指由于各种原因引起的肾功能突然下降 结构和功能受损。AKI在美国的患病率正在迅速上升，目前 这是全球医疗保健的负担。到目前为止，还没有有效的治疗方法来缓解促炎性疾病 以及损伤引起的纤维化表型。有趣的是，它最近得到了流行病学的支持 数据表明，女性性行为是AKI进展为CKD的保护因素。利用性别对比 缺血性损伤的特定机制具有确定新的、有效的治疗方法的难以置信的潜力 目标。因此，我将采用单核RNA和ATAC测序，以及空间测序 转录学为女性双侧脑缺血患者的损伤时间过程生成多模式图谱 再灌注损伤(BiIRI)。最近，在雄性小鼠中发现了一种新的细胞状态 单核RNA测序。这些细胞被称为修复失败的近端小管细胞(FR-PTC)，代表着 复苏道路上的死胡同，并可能对增殖、纤维化和 损伤后使肾组织恶化的炎症。这些FR-PTCs表现出差异表达的特征 来自健康和修复细胞状态的独特基因。这些遗传标记之一，它也是一种 与CKD相关的全基因组关联研究基因为Myh9。Myh9基因表达上调 尤其是在受损和失败的修复细胞团中，在健康和修复近端发现低水平 肾小管细胞。该基因编码非肌肉肌球蛋白IIA(NMIIA)，NMIIA对细胞形态起重要作用。 黏附、迁移和胞质分裂。它直接与肌动蛋白相互作用，形成稳定的细胞骨架网络。极端的 Myh9表达的变化可能转化为NMIIA丰度的变化，从而改变对 下游信号转导，有助于AKI的表型特征。因此，我假设性行为 Myh9在肾脏中的二态表达和NMIIA激活的性别特异性机制有助于 保护女性的肾功能。初步数据显示，雌性C57BL/6J小鼠在基线时有更高的 Myh9的表达高于雄性，这可能表明雌性已经形成了减少 NMIIA通过下调缺血反应激酶的活性。这项提案将调查这些 单核BiIRI后雌雄动物表达的时程变化 测序和空间转录。此外，缺血与NMIIA功能的关系将 在体内和体外细胞模型中使用金标准生化技术进行研究。归根结底，这 该提案将生成一份AKI性别差异的转录图谱，并阐明NMIIA在 AKI到CKD的疾病进展，潜在地确定了一个新的性别特异性治疗靶点。

ABSTRACT The cellular mechanisms that drive the progression of acute kidney injury (AKI) to chronic kidney disease (CKD) are not well understood. AKI is the abrupt decrease of kidney function from a variety of causes that elicits structural and functional damage. The prevalence of AKI is increasing rapidly in the United States and presents a healthcare burden worldwide. To date, there are no effective therapies for the mitigation of the proinflammatory and fibrotic phenotypes resulting from injury. Interestingly, it has been recently supported with epidemiological data that female sex is a protective factor in the AKI progression to CKD. Leveraging the comparison of sex specific mechanisms in ischemic injury has incredible potential for the identification of novel, effective therapeutic targets. Therefore, I will be employing single nuclei RNA and ATAC-Sequencing, as well as spatial transcriptomics to generate a multimodal atlas of the injury time course in females undergoing bilateral ischemia reperfusion injury (Bi-IRI). Recently, a novel cell state has been identified in male mice following Bi-IRI using single nuclei RNA sequencing. Known as failed repair proximal tubule cells (FR-PTC), these cells represent a dead end in the recovery pathway and could be lending a role to increases in proliferation, fibrosis, and inflammation that worsen kidney tissue after injury. These FR-PTCs exhibit a profile of differentially expressed genes that are unique from healthy and repairing cell states. One of these genetic markers, which is also a genome wide association study (GWAS) gene associated with CKD, was Myh9. Myh9 expression is upregulated specifically in injured and failed repair cell clusters, and is found in low levels in healthy and repairing proximal tubule cells. This gene encodes for nonmuscle myosin IIA (NMIIA), which plays an important role in cell shape, adhesion, migration, and cytokinesis. It interacts directly with actin, creating a stable cytoskeletal network. Drastic changes in Myh9 expression may translate into changes in NMIIA abundance that could alter the regulation of downstream signaling, contributing to phenotypes characteristic of AKI. Therefore, I hypothesize that sexually dimorphic expression of Myh9 in the kidney and sex-specific mechanisms of NMIIA activation contribute to the renoprotection of female sex. Preliminary data suggests that female C57BL/6J mice at baseline have higher expression of Myh9 than males, potentially indicating that females have developed mechanisms for decreasing activity of NMIIA through downregulation of ischemia responsive kinases. This proposal will investigate these expression changes in males and females along the injury time course following Bi-IRI using single nuclei sequencing and spatial transcriptomics. Additionally, the relationship between ischemia and NMIIA function will be investigated using gold standard biochemical techniques in in vivo and in vitro cell models. Ultimately, this proposal will generate a transcriptomic atlas of sex differences in AKI and illuminate the role of NMIIA in the disease progression of AKI to CKD, potentially identifying a new sex-specific therapeutic target.