Histone deacetylase 9 is an epigenetic suppressor of intrarenal angiotensinogen, serving as a key mechanism in angiotensinogen augmentation in hypertension

 描述(申请人提供)：增强的肾内血管紧张素原(AGT)是肾内肾素-血管紧张素系统(RAS)激活的关键贡献者，导致 高血压的发展及相关的组织损伤。因此，阐明AGT上调的潜在机制对于制定治疗这些疾病的策略至关重要。慢性Ang II升高导致肾脏免疫细胞浸润增加，导致肾内细胞因子水平升高，特别是白细胞介素6(IL-6)。值得注意的是，IL-6是血管紧张素转换酶II诱导的肾近端小管细胞(PTC)AGT增强所必需的。然而，这些复杂的相互作用导致AGT增强的细胞机制还没有被描绘出来。在正常情况下，雌性啮齿动物的肾皮质AGT水平低于雄性啮齿动物；然而，血浆和肝脏AGT水平没有这种性别差异，这表明肾脏具有建立基础AGT水平所固有的独特机制。我们的初步研究表明，雌性大鼠肾脏中组蛋白脱乙酰酶9(HDAC9)的水平高于雄性大鼠，并且小干扰RNA和一种抑制剂抑制HDAC9可导致PTC中AGT水平的升高。这些结果表明，HDAC9是肾内AGT表达的抑制因子。由于HDAC通过组蛋白去乙酰化抑制基因转录，HDAC水平的变化改变了激活的转录因子与其在基因启动子上的结合位置的可及性。初步研究表明，血管紧张素转换酶II可降低PTC患者的HDAC9水平。因此，我们假设血管紧张素转换酶II下调HDAC9可以减少HDAC9对AGT表达的抑制作用。这允许病理刺激激活的其他转录因子，特别是IL-6，增强AGT的表达，从而导致高血压的进展和血管紧张素Ⅱ依赖型高血压的相关组织损伤。为了解决这一假设，我们将利用独特的PTC和Ang II依赖的高血压动物模型(特定目标1)，在Ang II依赖高血压的发展过程中，建立Ang II抑制肾内HDAC9水平。此外，拟议的研究将证明，Ang II诱导的肾内HDAC9下调是通过Ang II和IL-6的协同作用增强肾内AGT并随后发展为高血压所必需的(特定目标2)。在这些实验中，通过siRNA敲除HDAC9和通过AAV2感染过表达HDAC9将被用来证明HDAC9敲除和IL-6协同增强PTC中AGT的表达，以及肾脏特异性HDAC9的过度表达减弱了肾内AGT的升高，从而促进了Ang II依赖型高血压肾脏损伤的发展。该项目还将调查肾内HDAC9调节的性别差异及其在高血压AGT调节中的作用。这些结果将确立肾内HDAC9在调节血管紧张素Ⅱ依赖型高血压肾内AGT中的主要作用，并为以HDAC9为靶点治疗高血压和RAS相关组织损伤提供机制基础。

 DESCRIPTION (provided by applicant): Augmented intrarenal angiotensinogen (AGT) is a critical contributor to the activation of intrarenal renin- angiotensin system (RAS) which leads to the development of hypertension and associated tissue injury. Therefore, elucidating mechanisms underlying AGT upregulation is essential for the development of strategies to treat these diseases. Chronic Ang II elevation induces increased immune cell infiltration in the kidneys contributing to increase in intrarenal cytokine levels specifically interleukin 6 (IL-6). Notably, IL-6 is required for Ang II-induced AGT augmentation in renal proximal tubular cells (PTC). However, the cellular mechanisms underlying these complex interactions leading to AGT augmentation have not been delineated. Renal cortical AGT levels are lower in female rodents than in males under normal conditions; however, plasma and liver AGT levels do not exhibit this gender disparity, suggesting that kidneys have a unique mechanism inherent to establishing basal AGT levels. Our preliminary studies demonstrated that histone deacetylase 9 (HDAC9) levels are higher in the kidneys of female rats than those of male rats and that HDAC9 inhibitions by siRNA and an inhibitor resulted in augmentation of AGT levels in PTC. These results indicate that HDAC9 is a suppressor of intrarenal AGT expression. Since HDACs suppress gene transcription by histone-deacetylation, changes in HDAC levels alter accessibility of activated transcription factors to their binding sites on gene promoters. The preliminary study showed that HDAC9 levels are decreased by Ang II in PTC. Accordingly, we hypothesize that downregulation of HDAC9 by Ang II reduces the suppressive effects of HDAC9 on AGT expression. This allows other transcription factors activated by pathological stimuli, in particula IL-6, to augment AGT expression, thus leading to the progression of hypertension and associated tissue injury in Ang II-dependent hypertension. To address this hypothesis, we will establish that Ang II suppresses intrarenal HDAC9 levels during the development of Ang II-dependent hypertension using unique PTC and Ang II- dependent hypertensive animal models (Specific Aim 1). In addition, the proposed studies will demonstrate that Ang II-induced intrarenal HDAC9 downregulation is required for intrarenal AGT augmentation by synergistic effects of Ang II and IL-6 and consequent development of hypertension (Specific Aim 2). In these experiments, HDAC9 knockdown by siRNA and overexpression via AAV2 infection will be used to show that the HDAC9 knockdown and IL-6 synergistically augment AGT expression in PTC and that kidney-specific HDAC9 overexpression attenuates intrarenal AGT elevation facilitating consequent the development of kidney injury in Ang II-dependent hypertension. Gender differences of intrarenal HDAC9 regulation and its contribution to AGT regulation in hypertension will also be investigated in the projects. The results will establish the cardinal rol of intrarenal HDAC9 in the regulation of intrarenal AGT in Ang II-dependent hypertension and provide a mechanistic rationale for targeting HDAC9 to treat hypertension and RAS associated tissue injury.