Urine biomarkers of lupus nephritis pathology and response to therapy

描述（由申请人提供）： 狼疮性肾炎（LN）导致高达42%的患者在五年后出现肾衰竭。然而，传统的生物标志物和治疗反应的临床指标通常不能检测治疗失败，直到对肾脏的不可逆损伤已经发生。因此，需要一种更可靠的方法来确定LN的诊断和对诱导治疗的反应。本项目的目标是为临床医生和研究人员提供1）用于诊断LN的国际肾脏病学会/肾脏病理学会（ISN/RPS）分类（Class）的非侵入性可靠工具，2）对治疗的长期反应的短期标志物，3）用于临床使用的生物标志物的经验证的测定，以及4）关于可用作未来治疗靶点的对治疗的反应机制的假设。在资助的前三年，生物标志物发现工作提供了初步数据，这些数据集中在有限数量的候选标志物上。反应性中间产物的标记物也显示出作为生物标记物的前景。我们的初步数据和途径分析表明，LN中候选生物标志物和氧化应激之间存在联系。在初步研究中，使用这组缩小的标志物的模型在诊断Class和预测对治疗的反应方面比传统的生物标志物或单一生物标志物模型具有更高的预测能力。我们推测，表达的变化和反应性中间体诱导的尿蛋白的翻译后修饰将作为LN的类和治疗反应的生物标志物。为了解决这些假设，提出了以下具体目的：1）创建并验证狼疮性肾炎（LN）中国际肾脏病学会/肾脏病理学会（ISN/RPS）活检分类（Class）的尿生物标志物诊断模型，2）创建并验证预测LN中对诱导治疗的肾反应的尿生物标志物模型，以及3）开发并验证用于临床使用的生物标志物测定。受试者将来自查尔斯顿和巴尔的摩初始队列以及Genentech LUNAR研究人群。将在入组时确定肾炎的ISN/RPS分类，并根据美国风湿病学会和系统性红斑狼疮国际合作临床肾反应标准描述诱导治疗开始后1年的结局。将在入组后0个月和3个月采集尿液。候选低丰度蛋白质（从第一个资助期的发现分析中选择的趋化因子、生长因子、细胞因子和肾损伤标志物）将通过Luminex微珠测定和ELISA进行定量。将通过高效液相色谱-电化学检测（HPLC-EC）检测指示暴露于反应性中间体的尿蛋白酪氨酸（Tyr）的翻译后修饰。将使用基线（针对目标1）和基线以及三个月（针对目标2）时的个体标志物水平创建数学模型（通过人工神经网络（ANN）），该模型使用替代尿蛋白标志物水平：1）诊断基线时的LN类别和2）预测一年时的反应治疗。由于机器学习的技术性质，需要熟悉ANN分析的评审员。在第三个目标中，将开发反应性中间体修饰Tyr的免疫测定法，所有生物标志物免疫测定法将进行适合正式生物标志物确认的方法验证。这后一步是必不可少的快速接受和应用本研究的结果，以临床。途径分析将用于探索在前两个目标中被认为具有预测性的生物标志物之间的机制相互作用。关于疾病活动，损伤和治疗反应的机制的假设将被确定，可以利用靶向治疗。这项研究的独特之处在于：1）用于训练和验证预测模型的群体的规模、多样性和严格的特征描述; 2）使用代表不同致病机制的新型且描述良好的生物标志物，以在单个模型中进行测试。长期目标是为临床医生提供一个合格的生物标志物面板加上一个基于网络的预测工具，更准确的诊断和治疗监测LN。 公共卫生相关性： 与退伍军人健康的相关性。狼疮肾炎影响着退伍军人中越来越多的妇女和有色人种妇女，少数民族和妇女之间的健康差距是退伍军人健康研究计划的优先事项。在狼疮性肾炎妇女中发现的不良结局标志物将通过提供非侵入性疾病标志物和短期治疗反应指标，为临床医生和研究人员提供药物开发和患者护理的增强和必要工具。本研究中使用的生物标志物鉴定技术广泛适用于其他肾脏疾病，如糖尿病和高血压，这两种疾病在退伍军人人群中普遍存在，并与不良结局相关。因此，这项研究的结果可以推进非侵入性肾脏监测进展进展的肾小球肾炎在一些疾病。

DESCRIPTION (provided by applicant): Lupus nephritis (LN) results in renal failure in up to 42% of patients after five years. However, traditional biomarkers and clinical indicators of treatment response often cannot detect treatment failure until irreversible damage to the kidneys has occurred. Therefore, a more reliable means of determining diagnosis and response to induction therapy in LN is needed. The goals of this project are to provide clinicians and investigators with 1) a non-invasive, reliable tool for diagnosis of International Society of Nephrology/Renal Pathology Society (ISN/RPS) class (Class) in LN, 2) a short-term marker of long-term response to therapy, 3) validated assays of biomarkers for clinical use, and 4) hypotheses about mechanisms of response to therapy that can be used as future therapeutic targets. During the first three years of funding, biomarker discovery efforts provided preliminary data that has focused efforts on a limited number of candidate markers. Markers of reactive intermediate production also showed promise as biomarkers. Links between candidate biomarkers and oxidative stress in LN were suggested by our preliminary data and pathways analysis. In preliminary studies, models using this narrowed set of markers had increased predictive power in diagnosing Class and predicting response to therapy over traditional biomarker or single biomarkers models. We hypothesize that changes in expression of and reactive intermediate-induced post-translational modifications of urinary proteins will serve as biomarkers of Class and response to therapy in LN. To address these hypotheses, the following Specific Aims are proposed: 1) Create and validate models of urine biomarkers diagnostic of International Society of Nephrology/Renal Pathology Society (ISN/RPS) biopsy class (Class) in lupus nephritis (LN), 2) Create and validate models of urine biomarkers predictive of renal response to induction therapy in LN, and 3) Develop and validate biomarker assays for clinical use. Subjects will derive from the Charleston and Baltimore inception cohorts and the Genentech LUNAR study population. ISN/RPS class of nephritis will be determined at entry, and outcomes at one year after start of induction therapy will be characterized by the American College of Rheumatology and Systemic Lupus Erythematosus International Cooperating Clinics renal response criteria. Urine will be collected at zero and three months after entry. Candidate low abundance proteins (chemokines, growth factors, cytokines, and renal damage markers selected from discovery analysis in the first funding period) will be quantitated by the Luminex bead assay and ELISA. Post-translational modifications of urine protein tyrosines (Tyr) indicative of exposure to reactive intermediates will be detected by high performance liquid chromatography-electrochemical detection (HPLC-EC). Levels of individual markers at baseline (for Aim 1) and baseline and three months (for Aim 2) will be used to create a mathematical models (by artificial neural network (ANN) that use the levels of surrogate urine protein markers to: 1) diagnose Class of LN at baseline and 2) predict response therapy at one year. Due to the technical nature of machine learning, a reviewer familiar with ANN analysis is requested. In the third Aim, an immunoassay for reactive-intermediate-modified Tyr will be developed, and all biomarker immunoassays will undergo method validation suitable for formal biomarker qualification. This latter step is essential to rapid acceptance and application of the findings of this study to the clinic. Pathways analysis will be used to explore mechanistic interactions between biomarkers deemed predictive in the first two aims. Hypotheses regarding mechanisms of disease activity, damage, and response to therapy will be identified that could be exploited with targeted therapies. This study is unique in 1) the size, diversity, and rigorous characterization of the population used to train and validate the predictive models and 2) its use of novel and well described biomarkers representing diverse pathogenic mechanisms to be tested in a single model. The long-term goal is to provide clinicians with a qualified biomarker panel coupled with a web-based predictive tool for more accurate diagnosis and therapeutic monitoring of LN. PUBLIC HEALTH RELEVANCE: Narrative-Relevance to Veterans Health. Lupus nephritis affects a growing population of women and women of color in the veteran population, and health disparities among minorities and women are a priority for Veterans Health research initiatives. The markers identified for poor outcomes in women with lupus nephritis will give clinicians and researchers enhanced and necessary tools for drug development and patient care by providing noninvasive markers of disease and short term therapeutic response indicators. The biomarker qualification techniques used in this study are broadly applicable to other renal diseases such as diabetes and hypertension, both of which are prevalent in the veteran population and are associated with poor outcomes. Thus, the results of this study could advance the progress of non-invasive renal monitoring for advancing glomerulonephritis in a number of diseases.