The role of Nox2 in CNI-induced renal fibrosis

描述(申请人提供)：尽管实体器官移植在过去的20年里取得了巨大的成功，但慢性钙调神经磷酸酶抑制剂(CNI)介导的纤维化限制了长期肾脏结果。CNIs包括环孢素A(CsA)和他克莫司(TAC)，是移植抗排斥治疗的基础。然而，它们代表了一个主要的公共卫生问题，因为CNI诱导的肾脏纤维化是实体器官移植后肾功能衰竭的重要原因。越来越多的证据表明，NOX2处于CNI诱导的肾脏缺氧和纤维化的十字路口。NOX2是典型的NADPH氧化酶的吞噬异构体，负责“氧化爆发”，即消除内在化病原体的机制。我们推测，与其在吞噬细胞中的作用无关，NOX2是CNI诱导的肾脏缺氧和纤维化的关键调节因子。为了支持这一假说，我们实验室的观察表明：(A)在CsA诱导的上皮向间充质转化(EMT)过程中，肾小管上皮细胞可诱导NOX2的表达；(B)在CsA诱导的纤维化过程中，肾小管间质NOX2表达上调；(C)通过血氧水平依赖的MRI(BOLD MRI)测量，Apoynin和DPI抑制NOx的活性与预防CsA诱导的纤维化和缺氧有关。基于这些观察，以及NOX2基因敲除小鼠的可用性来严格测试我们的假设，我们提出了以下研究。在特定的目标1中，我们将描述在CNI诱导的EMT和肾小管上皮细胞基质重塑过程中调控NOX2活性的分子机制。接下来，我们将确定NOX2在CNI诱导的肾纤维化中的具体作用。我们将使用野生型、NOX2、TGFb1和Smad2/3缺失的小鼠和肾小管细胞进行这些研究。在特定的目标2中，我们将使用BOLD和对比剂增强的非侵入性MR成像研究来表征慢性CsA治疗对小鼠延髓和皮质的灌注和氧合的影响。我们将用包括匹莫硝唑和HIF-1a在内的组织缺氧的分子标志物来证实这些研究。最后，我们将确定NOX2的缺失是否改善了CsA诱导的小鼠低灌流和低氧。在具体目标3中，我们将在20例非肾器官移植受者的试点临床研究中证实上述分子和小动物研究的有效性。这项研究旨在确定肾内NOX2是否与慢性CNI肾毒性有关。第二个目标是确定通过BOLD MRI测量的肾内氧合是否可以预测疾病的进展。综上所述，我们概述了严格评估NOX2在CNI诱导的肾纤维化中的作用的研究。此外，将提出当代和补充的临床和翻译策略，以研究在CNI介导的纤维化形成过程中调节NOX2活性的分子机制。如果成功，我们的研究结果将在设计新的诊断、监测和治疗方案方面向前迈出重要的一步，旨在抵消免疫抑制疗法的有害影响，并改善长期肾脏结果。 公共卫生相关性：钙调神经磷酸酶抑制剂引起的慢性肾功能衰竭是器官移植受者一个重要的公共卫生问题。我们建议在体外、动物和人体内进行研究，考察NOX2作为肾脏纤维化介质的具体作用。我们还提出了非侵入性成像技术来诊断和监测这些药物造成的肾脏瘢痕。如果成功，这些研究可能导致制定治疗目标和诊断/监测方法，以预防器官移植受者的肾脏纤维化。

DESCRIPTION (provided by applicant): Despite the tremendous success of solid organ transplantation in past 20 years, long-term kidney outcomes have been limited by chronic calcineurin inhibitor (CNI)-mediated fibrosis. CNIs including cyclosporine A (CsA) and tacrolimus (TAC) are the backbone of anti-rejection therapy in transplantation. However, they represent a major public health concern since CNI-induced kidney fibrosis is an important cause of renal failure after solid organ transplantation. Increasing evidence suggests that Nox2 is at the crossroads of CNI-induced renal hypoxia and fibrosis. Nox2 is the classical phagocytic isoform of the NADPH oxidase enzyme responsible for "oxidative burst", the mechanism that eliminates internalized pathogens. We hypothesize that independent from its role in phagocytes, Nox2 is a key regulator of CNI-induced kidney hypoxia and fibrosis. In support of this hypothesis, observations from our laboratory demonstrate that (a) Nox2 is inducible in renal tubular epithelial cells during CsA-induced epithelial-to-mesenchymal transition (EMT) (b) tubulointerstitial Nox2 is upregulated in kidneys undergoing CsA-induced fibrosis (c) inhibition of Nox activity with apocynin and diphenyleneiodonium (DPI) is associated with prevention of CsA-induced fibrosis and hypoxia measured by blood oxygen-level-dependent MRI (BOLD MRI) in rats. Based on these observations, and the availability of Nox2 knockout mice to rigorously test our hypotheses, we propose the following studies. In Specific Aim 1, we will characterize the molecular mechanisms that regulate Nox2 activity during CNI-induced EMT and matrix remodeling in renal tubular epithelial cells. Next, we will define the specific role of Nox2 in CNI-induced renal fibrosis. We will perform these studies using wild type, Nox2, TGFb1 and Smad2/3-null mice and tubular cells. In Specific Aim 2, we will characterize the effects of chronic CsA therapy on medullary and cortical perfusion and oxygenation using noninvasive MR-imaging with BOLD and contrast-enhanced perfusion studies in mice. We will confirm these studies with molecular markers of tissue hypoxia including pimonidazole and HIF-1a. Last, we will determine if the absence of Nox2 improves CsA-induced hypoperfusion and hypoxia in mice. In Specific Aim 3, we will confirm the validity of the above molecular and small animal investigations in a pilot clinical study of 20 nonkidney organ transplant recipients. The study is designed to determine whether intrarenal Nox2 is associated with chronic CNI nephrotoxicity. The secondary objective is to determine whether intrarenal oxygenation measured by BOLD MRI can predict disease progression. In summary, we outline research studies that rigorously assess the role of Nox2 in CNI-induced renal fibrosis. In addition, will propose contemporary and complementary clinical and translational strategies to examine the molecular mechanisms that regulate Nox2 activity during CNI-mediated fibrogenesis. If successful, the results of our studies will provide a significant step forward in the design of new diagnostic, monitoring and treatment options aimed to offset the deleterious effects of immunosuppressant therapy and improve long-term kidney outcomes. PUBLIC HEALTH RELEVANCE: Chronic kidney failure from calcineurin inhibitors is an important public health concern in organ transplant recipients. We propose in vitro, animal and human studies that examine the specific role of Nox2 as a mediator of kidney fibrosis. We also propose noninvasive imaging technologies to diagnose and monitor renal scarring from these drugs. If successful these studies could lead to the development of treatment targets and diagnostic/monitoring methods for the prevention of kidney fibrosis in organ transplant recipients.