The role of Nox2 in CNI-induced renal fibrosis

描述（申请人提供）：尽管在过去20年中实体器官移植取得了巨大成功，但慢性钙调磷酸酶抑制剂（CNI）介导的纤维化限制了肾脏的长期预后。包括环孢素A （CsA）和他克莫司（TAC）在内的CNIs是移植抗排斥治疗的支柱。然而，它们代表了一个主要的公共卫生问题，因为cni诱导的肾纤维化是实体器官移植后肾功能衰竭的一个重要原因。越来越多的证据表明，Nox2处于cni诱导的肾缺氧和纤维化的十字路口。Nox2是NADPH氧化酶的经典吞噬异构体，负责“氧化爆发”，这是消除内化病原体的机制。我们推测，除了在吞噬细胞中的作用外，Nox2是cni诱导的肾缺氧和纤维化的关键调节因子。为了支持这一假设，我们实验室的观察表明(a)在csa诱导的上皮-间质转化（EMT）期间，Nox2在肾小管上皮细胞中被诱导；(b)在csa诱导的纤维化过程中，肾小管间质Nox2在肾脏中被调高；(c)罗布酚和二苯基碘（DPI）抑制Nox活性与大鼠血氧水平依赖MRI （BOLD MRI）测量的csa诱导的纤维化和缺氧的预防有关。基于这些观察结果，以及Nox2敲除小鼠的可用性来严格验证我们的假设，我们提出以下研究。在Specific Aim 1中，我们将描述在cni诱导的肾小管上皮细胞EMT和基质重塑过程中调节Nox2活性的分子机制。接下来，我们将明确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-null小鼠和小管细胞进行这些研究。在特异性目标2中，我们将利用无创磁共振成像和对比增强灌注研究来表征慢性CsA治疗对小鼠髓质和皮质灌注和氧合的影响。我们将用组织缺氧的分子标记包括吡咪唑和HIF-1a来证实这些研究。最后，我们将确定Nox2的缺失是否能改善csa诱导的小鼠低灌注和缺氧。在Specific Aim 3中，我们将在20例非肾器官移植受者的初步临床研究中证实上述分子和小动物研究的有效性。该研究旨在确定肾内Nox2是否与慢性CNI肾毒性有关。次要目的是确定BOLD MRI测量的肾内氧合是否可以预测疾病进展。总之，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，将提出当代和互补的临床和翻译策略，以检查在cni介导的纤维形成过程中调节Nox2活性的分子机制。如果成功，我们的研究结果将为设计新的诊断、监测和治疗方案提供重要的一步，旨在抵消免疫抑制剂治疗的有害影响，改善长期肾脏预后。

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.