The Role of Angiotensin II on mTOR mediated Tubulointerstitial Fibrosis

描述(申请人提供)：我们的建议旨在研究血管紧张素II对肾脏老化和肾小管间质纤维化的影响，这是一个与老年进行性肾脏疾病相关的机制。肾小管间质纤维化是预测临床肾脏疾病进展的主要组织学发现。有足够的数据支持阻断血管紧张素II 1型受体(AT1R)可以改善肾小管间质纤维化和肾脏疾病的进展；然而，尽管广泛使用AT1R阻断，CKD和终末期肾病的负担在我们的老年人中仍然很高。这凸显了研究预防进行性慢性肾脏病的策略的必要性。在这种背景下，靶向降低哺乳动物雷帕霉素靶标(MTOR)/S6K1活性以改善多囊肾病和糖尿病肾病模型的肾小管间质纤维化和肾功能一直受到极大的关注。MTOR在营养/压力感知通路中具有战略地位，这些通路有助于TOR/S6K1功能在调节慢性病和衰老方面的重要性。 我们的初步数据表明，Ang II直接参与了mTOR和S6K1的激活，导致了肾小管间质纤维化。我们的数据进一步表明，这种机制是通过丢失黏附分子，如近端小管特异性N-钙粘附素和促进上皮-间充质转化(EMT)来实现的。钙粘附素丢失是通过EMT治疗肾小管间质纤维化的关键第一步。在肾小管间质纤维化和进行性肾脏疾病中，尤其是在衰老的情况下，对激动剂引起的EMT和粘连丧失的了解有限。 因此，这项临床前建议广泛地用于研究一种新的机制，该机制通过具有直接临床相关性的干预措施来服务于重要的临床需求领域。我们的目标是针对早期肾小管间质纤维化，以防止衰老过程中的进行性肾脏疾病。在我们提出的工作中，在特定的目标I中，我们将研究血管紧张素II激活mTOR/S6K1和近曲小管特异性黏附分子N-钙粘附素的丢失在进展性肾脏疾病模型中与EMT和肾小管间质纤维化的关系。然后，我们将通过组织形态学和免疫组织化学分析，以及通过透射电子显微镜的超微结构分析，探讨mTOR和S6K1的激活/磷酸化以及N-钙粘素在早期肾小管间质纤维化中表达缺失的新作用。为了进一步探讨mTOR/S6K1在N-钙粘蛋白丢失中的具体作用，在特定的目标2中，我们将重点利用RNA沉默的击倒策略来描述近端小管中决定N-钙粘蛋白丢失的特定复合体。我们提出了在原代培养的衰老近端小管细胞中AT1R、mTORC1/S6K1的RNAi，并结合EMT和纤维化的特异性标志物来检测N-钙粘附素。 公共卫生相关性：老龄化是肾脏疾病的主要风险因素，慢性肾脏疾病(CKD)和终末期肾脏疾病(ESRD)的护理是美国主要的医疗支出之一。肾小管间质纤维化是预测肾脏疾病进展的主要发现。然而，我们对衰老过程中导致纤维化和进行性肾脏疾病的机制的了解仍然有限。这突显了需要研究在早期阶段中断这一机制的策略，以防止人口老龄化中的进行性慢性肾脏病。因此，我们的建议试图通过具有直接临床相关性的干预方案来研究一种有助于肾小管间质纤维化早期发展的新机制。

DESCRIPTION (provided by applicant): Our proposal seeks to investigate the impact of angiotensin (Ang) II on the aging kidney and tubulointerstitial fibrosis, a mechanism that correlates with progressive kidney disease in aging. Tubulointerstitial fibrosis is the predominant histomorphologic finding that predicts progression of clinical kidney disease. Sufficient data exist to support that blockade of the Ang II type 1 receptor (AT1R) has been shown to improve tubulointerstitial fibrosis and progression of kidney disease; however, the burden of CKD and ESRD remains high in our elderly despite widespread use of AT1R blockade. This highlights the need to investigate strategies to prevent progressive CKD. In this context, there has been tremendous interest in targeting reductions in mammalian Target of Rapamycin (mTOR)/S6K1 activity to improve tubulointerstitial fibrosis and kidney function in polycystic and diabetic kidney disease models. mTOR holds a strategic position at the nexus of nutrient/stress sensing pathways that contribute to the importance of TOR/S6K1 function in regulating chronic disease and in aging. Our preliminary data suggest that Ang II contributes directly to activation of mTOR and S6K1 resulting in tubulointerstitial fibrosis. Our data further suggest the mechanism occurs through loss of adhesion molecules such as the proximal tubule specific N-cadherin and promotion of epithelial-mesynchymal transition (EMT). Cadherin loss is a critical first step in tubulointerstitial fibrosis through EMT. Understanding of agonists that elicit EMT and loss of adhesion are limited in tubulointerstitial fibrosis and progressive kidney disease, especially in the context of aging. Thereby, this preclinical proposal broadly serves to investigate a novel mechanism that serves a significant area of clinical need with interventions that have direct clinical relevance. Our aims have been developed to target early tubulointerstitial fibrosis in order to prevent progressive kidney disease in aging. In our work proposed, in Specific Aim I we will examine the role of Ang II activation of mTOR/S6K1 and loss of the proximal tubule specific adhesion molecule N-cadherin in relation to EMT and tubulointerstitial fibrosis in progressive kidney disease in an aging rodent model. We will then explore activation/phosphorylation of mTOR and S6K1 and the novel role for loss of N-cadherin expression in early stage tubulointerstitial fibrosis on histomorphologic and immunohistochemical analysis as well as EMT via ultrastructural analysis with transmission electron microscopy. To further explore the specific role of mTOR/S6K1 roles on N-cadherin loss, in Specific Aim 2 we will focus on utilization of knockdown strategies with RNA silencing to delineate the specific complex within the proximal tubule that dictates loss of N-cadherin. We propose RNAi of AT1R, mTORC1/S6K1 in primary culture of aging proximal tubule cells and measure N-cadherin in conjunction with specific markers of EMT and fibrosis. PUBLIC HEALTH RELEVANCE: Aging is a leading risk factor for kidney disease and care for chronic kidney disease (CKD) and end-stage renal disease (ESRD) is one of the leading healthcare expenditures in the US. Tubulointerstitial fibrosis is the predominant finding that predicts progression of kidney disease. However, our understanding of mechanisms that elicit fibrosis and progressive kidney disease in aging remain limited. This highlights the need to investigate strategies that interrupt this mechanism at early stages to prevent progressive CKD in the aging population. Thereby, our proposal seeks to investigate a novel mechanism that contributes to the early development of tubulointerstitial fibrosis with interruptions schemes that have direct clinical relevance.