Intratubular Angiotensin II and AT1a Receptors in The Proximal Tubules: Roles in Hypertension and Kidney Injury

高血压是心血管疾病、中风和终末期肾功能衰竭的最重要的危险因素。在美国 在美国，近46%的成年人患有高血压，并将在他们的一生中接受降压药治疗。仅限 50%的高血压患者对当前的降压药有反应，而三分之一的高血压患者仍然患有高血压 患者会出现心血管、中风和肾脏并发症。负责发展的机制 心血管和肾脏损伤以及对当前降压治疗反应差的原因仍然存在 不完全理解。因此，有必要进行进一步的研究，以揭示新的机制、途径和 未控制的高血压和靶器官损伤的治疗靶点。在初步研究中，我们使用了最先进的 SGLT2-Cre/loxP方法删除血管紧张素II(Ang II)AT1a受体、Na+/H+交换器3(NHE3)或密钥 线粒体蛋白sirtuin3(SIRT3)选择性地位于肾脏近端小管的S1和S2段。我们 有证据表明，近端小管特异性AT1a或NHE3的缺失降低了基础血压，增加了 压力钠尿反应，并减轻血管紧张素转换酶II诱导的高血压，以及近端小管特异性缺失 的AT1a受体显著减弱，而近端小管特异性SIRT3的缺失显著 加重肾缺血再灌注(I/R)损伤。这些初步研究强烈表明肾小管内血管 II及其AT1a受体和近曲小管SIRT3在高血压和高血压的发生发展中起重要作用 肾I/R损伤。在这项提议中，我们将检验肾小管内Ang II和AT1a受体在肾小管上皮细胞 肾近端小管在血管紧张素Ⅱ诱导的高血压和肾I/R的发展中是必需的 损伤，以及近端小管AT1a受体或血管紧张素原(AGT)选择性缺失将 减轻血管紧张素II诱导的高血压和肾脏I/R损伤有两个特定的目的。《特定目标1》将测试 近端小管Ang II和AT1a受体在维持基础血中起关键作用的假说 Na~+/H~+逆向转运体激活与血管紧张素Ⅱ诱导的高血压的发生发展 (NHE3)，钠和葡萄糖共转运体2(SGLT2)，以及对压力钠尿反应的调节。具体目标2 将检验近端小管AT1a受体在肾I/R损伤发病机制中起关键作用的假设， 由肾小管内和细胞内Ang II激活上调Toll样受体4(TLR-4)，下调 线粒体SIRT3的表达，并损害近端小管的线粒体功能。这些假设将 使用高度创新的小鼠模型进行测试，该模型具有全局和近端小管特异的a)AT1a基因敲除 受体；b)血管紧张素原；c)NHE3；d)SGLT2；e)TLR4；或f)SIRT3。血管紧张素Ⅱ依赖型高血压和 肾I/R损伤将由a)输注天然血管紧张素II引起；b)2肾、1夹肾性高血压；c)遗传性 编码的循环弹性蛋白样多肽-血管紧张素II(ELP-Ang II)；或d)过表达近端小管- 特异的线粒体靶向细胞内ECFP/Ang II.遥测血压，压力钠尿 无创肾小球滤过率、活体双光子成像和XFe24细胞外通量分析仪对 测量线粒体功能、电子显微镜和免疫组织化学成像以及蛋白质印迹分析 将对促炎症、促纤维化和信号蛋白进行研究。拟议的研究具有很高的 重大的和临床相关的，新的知识将导致对 高血压和肾I/R损伤的发病机制，并有助于开发近端小管靶向药物以治疗不良 控制性高血压和肾I/R损伤。

Hypertension is the most important risk factor for cardiovascular diseases, stroke, and end-stage kidney failure. In the United States, nearly 46% of adults develop hypertension and will be treated with antihypertensive drugs in their lifetime. Only 50% of hypertensive patients are responsive to current antihypertensive drugs, whereas 1/3 of remaining hypertensive patients will develop cardiovascular, stroke and renal complications. The mechanisms responsible for the development of cardiovascular and kidney injury and the reasons for poor responses to current antihypertensive therapies remain incompletely understood. Thus, further studies are necessary in order to uncover new mechanisms, pathways, and therapeutic targets of uncontrolled hypertension and target organ injury. In preliminary studies, we used the state of the art SGLT2-Cre/LoxP approach to delete angiotensin II (ANG II) AT1a receptors, the Na+/H+ exchanger 3 (NHE3), or a key mitochondrial protein sirtuin 3 (SIRT3) selectively in the S1 and S2 segments of the proximal tubules in the kidney. We have evidence that proximal tubule-specific deletion of AT1a or NHE3 decreases basal blood pressure, augments the pressure natriuresis response, and attenuates ANG II-induced hypertension, and that proximal tubule-specific deletion of AT1a receptors significantly attenuated, whereas proximal tubule-specific deletion of SIRT3 significantly worsened renal ischemia and reperfusion (I/R) injury. These preliminary studies strongly suggest that intratubular ANG II and its AT1a receptors and SIRT3 in the proximal tubules play an important role in the development of hypertension and renal I/R injury. In this proposal, we will test the hypotheses that intratubular ANG II and AT1a receptors in the proximal tubules of the kidney are required for the development of ANG II-induced hypertension and renal I/R injury, and that deletion of AT1a receptors or angiotensinogen (AGT) selectively in the proximal tubules will attenuate ANG II-induced hypertension and renal I/R injury in two specific aims. Specific Aim 1 will test the hypothesis that intratubular ANG II and AT1a receptors in the proximal tubules play a key role in maintaining basal blood pressure homeostasis and the development of ANG II-induced hypertension, via the activation of the Na+/H+ antiporter (NHE3), Na+ and glucose cotransporter 2 (sglt2), and the regulation of the pressure natriuresis response. Specific Aim 2 will test the hypothesis that AT1a receptors in the proximal tubules play a key role in the pathogenesis of renal I/R injury, activated by intratubular and intracellular ANG II to upregulate Toll-Like receptor 4 (TLR-4), downregulate mitochondrial SIRT3 expression, and impair mitochondrial function in the proximal tubules. These hypotheses will be tested using highly innovative mouse models with global and proximal tubule-specific knockout of a) AT1a receptors; b) angiotensinogen; c) NHE3; d) SGLT2; e) TLR4; or f) SIRT3. ANG II-dependent hypertension and renal I/R injury will be induced by a) infusing a native ANG II; b) 2-kidney, 1-clip renal hypertension; c) a genetically encoded circulating Elastin-Like Polypeptide-ANG II (ELP-ANG II); or d) overexpressing a proximal tubule- specific, mitochondria-targeting intracellular ECFP/ANG II. Telemetry blood pressure, the pressure natriuresis response, noninvasive glomerular filtration rate, intravital two-photon imaging and XFe24 Extracellular Flux Analyzer to measure mitochondrial function, electron microscopic and immunohistochemical imaging, and Western blot analyses of proinflammatory, profibrotic and signaling proteins will be studied. The proposed studies are highly significant and clinically relevant, and the new knowledge will lead to a paradigm shift on understanding the pathogenesis of hypertension and renal I/R injury, and help develop proximal tubule-targeting drugs to treat poorly controlled hypertension and renal I/R injury.