Role of Adipocyte Na/K-ATPase Signaling in the Development and Progression of Uremic Cardiomyopathy By Altering Adipocyte Phenotype

氧化应激在心血管疾病病理生理学中的作用一直是人们研究的主题。 相当感兴趣。虽然终末期肾病比较少见，但慢性肾脏病变程度较轻 疾病[CKD]，如CKD 3是相当常见的，并被认为是 心血管发病率和死亡率。氧化应激在肾功能衰竭的发展中起着关键作用。 相关的心肌病(也称为尿毒症心肌病)，在实验和临床上都是如此。 我们的研究小组发现，钠钾泵“Na/K-ATPase”可以影响细胞信号转导 通过“Na/K-ATPase信号”放大氧化应激。我们已经特别证明了这条途径 对包括肥胖/代谢综合征在内的几种疾病实验模型的病理生理学至关重要 和实验性尿毒症心肌病。我们和其他人也观察到脂肪细胞本身是一种 肥胖/代谢综合征模型中氧化应激的重要来源及其直接相关的介质 这些脂肪细胞的细胞表型在相关的心血管疾病中起着因果作用 肥胖/代谢综合征。这使我们相信脂肪细胞可能在尿毒症中发挥核心作用。 心肌病。因此，我们假设脂肪细胞通过Na/K-产生系统性氧化应激。 ATPase前馈氧化剂扩增环在尿毒症心肌病中的作用 这种情况。我们的团队已经从α1亚单位的N结构域中开发出一种细胞活性多肽，NaKtid 抑制Na/K-ATPase-ROS扩增。我们的初步结果显示， 专门针对脂肪细胞的NaKide还可以减轻氧化应激和炎性细胞因子 改善新陈代谢参数。我们的实验方法包括研究以确定 脂肪细胞Na/K-ATPase信号转导在实验性尿毒症心肌病发生发展中的作用 肾切除(PNX)小鼠模型(目标1)。我们还将把目标对准北极星 采用“慢病毒基因转移”策略(目的1)确定脂肪细胞来源的Na/K- ATPase/Src信号在尿毒症心肌病进展中的作用为了测试NaKide的偏离目标的效果，我们 还将使用带有组织特异性启动子的慢病毒载体，包括心脏和肾脏靶向NaKide 尤其是在这些组织中。此外，我们将使用慢病毒载体将c-Src-shRNA靶向脂肪细胞，作为一种 抑制Na/K-ATPase信号的替代策略，以证明c-Src作为下游信号转导通路的作用 Na/K-ATPase在加重氧化应激和最终尿毒症心肌病中的中介作用。在目标2中，我们 将采用从Sham或PNX操作的C57BL6小鼠和PNX操作的C57BL6小鼠和 进行RNAseq分析以研究与尿毒症相关的脂肪细胞表型改变和途径 心肌病表型。我们还将使用从C57BL6和Na/K-ATPaseα1+/-分离的原代脂肪细胞 将暴露于尿毒症毒素的小鼠，随后激活Na/K-ATPase信号，以确定 体外激活特定的下游分子通路，模拟体内的结果。这些实验 将使我们能够确定Na/K-ATPase信号和/或脂肪细胞是否是潜在的疾病靶点 干预。这些研究如果得到证实，可能会为未来改善尿毒症的研究提供基础。 心肌病表型。

The role of oxidant stress in the pathophysiology of cardiovascular disease has long been a subject of considerable interest. Although end stage renal disease is relatively uncommon, milder degrees of chronic kidney disease [CKD] such as CKD 3 are quite common and have been implicated as important determinant in cardiovascular morbidity and mortality. Oxidant stress plays a key role in the development of renal-failure- associated cardiomyopathy (also known as uremic cardiomyopathy), both experimentally and clinically. The “Na/K-ATPase,” a sodium-potassium pump, has been shown by our group to affect cellular signaling via the “Na/K-ATPase signaling,” which amplifies oxidative stress. We have specifically shown that this pathway is critical to the pathophysiology of several experimental models of disease including obesity/metabolic syndrome and experimental uremic cardiomyopathy. We and others have also observed that the adipocyte itself is an important source of oxidant stress in models with obesity/metabolic syndrome and that mediators directly tied to the cellular phenotype of these adipocytes play a causal role in the cardiovascular conditions associated with obesity/metabolic syndrome. This led us to believe that adipocytes could play a central role in uremic cardiomyopathy. Therefore, we hypothesize that adipocytes create systemic oxidant stress through the Na/K- ATPase feed-forward oxidant amplification loop in uremic cardiomyopathy and serve as a therapeutic target for this condition. Our group has developed a cell permeant peptide, NaKtide, from the N domain of the α1 subunit of the Na/K-ATPase, which inhibits Na/K-ATPase-ROS amplification. Our preliminary results show that the NaKtide, targeted specifically to adipocyte, attenuates oxidative stress and inflammatory cytokines, in addition to improving metabolic parameters. Our experimental approach includes studies to determine the role of adipocyte Na/K-ATPase signaling in the development of experimental uremic cardiomyopathy using a partial nephrectomy (PNx) mouse model with and without dietary manipulations (Aim 1). We will also target the NaKtide to the adipocyte using “lentiviral gene transfer” strategy (Aim 1) to determine the role of adipocyte-derived Na/K- ATPase/Src signaling in the progression of uremic cardiomyopathy. To test the off-target effects of NaKtide, we will also use lentiviral vectors with tissue specific promoters, including heart and kidney to targeted NaKtide specially in these tissues. Further, we will target c-Src-shRNA to adipocytes using lentivirus vector, as an alternate strategy of inhibiting Na/K-ATPase signaling, to demonstrate the role of c-Src as a downstream mediator of Na/K-ATPase in exacerbating oxidative stress and eventually uremic cardiomyopathy. In Aim 2, we will employ in vitro protocols for primary adipocytes, isolated from Sham or PNx operated C57BL6 mice and perform RNASeq analysis to study the adipocyte phenotypic alterations and pathways associated with uremic cardiomyopathy phenotype. We will also use primary adipocytes isolated from C57BL6 and Na/K-ATPase α1+/- mice which will be exposed to uremic toxins, subsequently activating Na/K-ATPase signaling, to determine the activation of specific downstream molecular pathways in vitro that mimic in vivo outcomes. These experiments will allow us to determine if the Na/K-ATPase signaling and/or adipocytes are potential targets for disease intervention. These studies if proven may provide a basis for the future studies to ameliorate uremic cardiomyopathy phenotype.