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%的患者在五年后发生肾功能衰竭。然而，在肾脏发生不可逆转的损害之前，传统的生物标志物和治疗反应的临床指标往往无法检测到治疗失败。因此，需要一种更可靠的方法来确定狼疮性肾炎的诊断和诱导治疗的反应。该项目的目标是为临床医生和研究人员提供1)国际肾脏病学会/肾脏病理学会(ISN/RPS)LN类别(CLASS)的非侵入性、可靠的诊断工具，2)治疗长期反应的短期标记物，3)临床使用的有效生物标记物分析，以及4)可用作未来治疗靶点的治疗反应机制假说。在资助的头三年里，生物标记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记物上。反应性中间产物的标志物也显示出作为生物标志物的前景。我们的初步数据和通路分析表明，候选生物标记物与LN的氧化应激之间存在联系。在初步研究中，与传统的生物标记物或单一生物标记物模型相比，使用这组缩小的标记物的模型在诊断Class和预测治疗反应方面具有更高的预测能力。我们假设，尿蛋白表达的变化和反应性中间体诱导的翻译后修饰将作为LN治疗类别和反应的生物标记物。为了解决这些假设，提出了以下具体目标：1)建立和验证国际肾脏学会/肾脏病理学会(ISN/RPS)狼疮性肾炎(LN)活检班(CLASS)尿液生物标记物诊断模型，2)建立和验证预测狼疮性肾炎(LN)肾脏诱导治疗反应的尿液生物标志物模型，以及3)开发和验证临床使用的生物标记物分析方法。受试者将来自查尔斯顿和巴尔的摩的初始队列以及基因泰克月球研究人群。ISN/RPS级肾炎将在入选时确定，诱导治疗开始后一年的结果将以美国风湿病学会和系统性红斑狼疮国际合作诊所肾脏反应标准为特征。尿液将在入院后0个月和3个月收集。候选低丰度蛋白(趋化因子、生长因子、细胞因子和肾损伤标记物从第一个资助期的发现分析中挑选出来)将通过Luminex珠法和ELISA进行定量。指示暴露于活性中间体的尿蛋白酪氨酸(Tyr)的翻译后修饰将通过高效液相色谱-电化学检测(HPLC-EC)进行检测。基线(目标1)、基线和3个月(目标2)的个体标志物水平将被用来创建数学模型(通过人工神经网络(ANN)，该模型使用替代尿蛋白标志物的水平来：1)基线诊断LN分级；2)预测一年后的应答治疗。由于机器学习的技术性质，需要一位熟悉人工神经网络分析的评审员。在第三个目标中，将开发一种针对反应中间体修饰的Tyr的免疫分析，所有生物标记物免疫分析方法都将经过适合于正式生物标记物鉴定的方法验证。后一步对于迅速接受这项研究的结果并将其应用于临床是至关重要的。通路分析将被用来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械相互作用。关于疾病活动、损害和治疗反应机制的假说将被确定，可用于靶向治疗。这项研究的独特之处在于：1)用于训练和验证预测模型的人群的大小、多样性和严格的特征；2)使用代表不同致病机制的新颖和描述良好的生物标记物在单一模型中进行测试。长期目标是为临床医生提供一个合格的生物标记物面板和一个基于网络的预测工具，以实现对LN的更准确的诊断和治疗监测。 公共卫生相关性： 叙事-与退伍军人健康的相关性。狼疮性肾炎影响着退伍军人人口中越来越多的妇女和有色人种妇女，少数族裔和妇女之间的健康差距是退伍军人健康研究倡议的优先事项。狼疮性肾炎妇女预后不良的标志物将通过提供疾病的非侵入性标志物和短期治疗反应指标，为临床医生和研究人员提供更强大和必要的药物开发和患者护理工具。这项研究中使用的生物标记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中普遍存在，且与不良预后相关。因此，这项研究的结果可能会推动非侵入性肾脏监测在一些疾病中进展为进展的肾小球肾炎。

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.