Identifying urinary biomarkers for early type 2 diabetic nephropathy

应聘者：玛丽亚姆·阿夫卡里安在圣路易斯华盛顿大学获得医学博士学位，并在纽约长老会医院-威尔康奈尔医学中心接受内科住院医师培训。她目前是马萨诸塞州综合医院和布里格姆妇女医院联合项目的肾病学研究员。从2010年7月开始，她将被任命为马萨诸塞州综合医院肾病科的讲师。导师：Ravi Thadhani博士是一位国际公认的临床和翻译研究员，在指导新研究员方面有着良好的记录。他现在是，也将继续是一位密切参与的导师，致力于让阿夫卡里安博士的培训、她提议的学习和职业发展取得成功。 研究：糖尿病肾病是终末期肾病的主要原因。然而，按照目前的护理标准，我们只能延缓这种疾病的发展。这部分是由于缺乏有效的治疗方法，部分是因为在肾损害严重或不可逆转之前，缺乏可靠的检测方法来诊断早期疾病。迫切需要新的诊断和治疗工具，这两种工具都需要更好地了解疾病的自然历史，即确定新的发病和进展标记。尿液的比较蛋白质组学分析(研究尿液蛋白成分及其在疾病状态下如何变化)已被用于确定几种肾脏疾病的标志物。塔哈尼博士(Mentor)的S团队此前曾使用这种方法识别多肽签名，该签名可在临床诊断前10年检测到早期糖尿病肾损伤的迹象。这项建议提出了一种多阶段的方法来识别早期糖尿病肾病中改变的蛋白质。初步数据描述了一种敏感的蛋白质组技术(iTRAQ：用于相对和绝对定量的等量标签)的校准和适应应用于尿液。在目标1A中，iTRAQ将用于比较患有和不患有糖尿病肾病的患者(分别为病例和对照)的尿样。这一无假说和无偏见的比较分析将产生早期糖尿病肾病候选生物标志物的初步列表。在目标1B中，这份清单将补充假设驱动的候选者，即那些有重要证据参与糖尿病肾病的人。例如，蛋白质(A)是糖尿病肾病相关通路的一部分，(B)在糖尿病肾病中改变了mRNA的表达，或(C)在糖尿病肾病中改变了蛋白质水平。这一步骤旨在将目前所有可用的信息纳入到这一生物标记物发现工作中。在目标3中，使用靶标特异性和定量分析，例如酶联免疫吸附试验，在独立队列中重新评估来自目标1A和1B的假定生物标记物的表达。这一步骤将有助于剔除假阳性，并识别其差异表达在DN中得到证实的标记的子集。在目标4中，这些已确认的标志物的表达将在疾病过程中表现出特征。该建议旨在生成一组早期糖尿病肾病的候选生物标志物，这些标志物已在原始研究人群中得到验证。未来的一步是在更广泛、更多样化的人群中测试这些候选人。从长远来看，我们希望经过验证的生物标志物将成为早期糖尿病肾病的一种新的诊断工具，并有助于揭示疾病的病理生理。 公共卫生相关性：在美国，糖尿病是终末期肾病和透析的主要原因。目前的医学测试只能在已经发生重大的、有时是不可逆转的损害后才能诊断这种疾病。我们计划寻找尿液标记蛋白，可以用来检测糖尿病对肾脏损害的早期迹象，当这种损害更有可能被阻止或逆转时。

DESCRIPTION (provided by applicant): Candidate: Maryam Afkarian received an MD-PhD from Washington University in St. Louis and underwent residency training in Internal Medicine at New York Presbyterian Hospital-Weill Cornell Medical Center. She is currently a Nephrology Research Fellow at the joint program of Massachusetts General Hospital and Brigham and Women's Hospital. Starting July 2010, she will be appointed as an instructor at the Nephrology Division of Massachusetts General Hospital. Mentor: Dr. Ravi Thadhani is an internationally recognized clinical and translational investigator with a strong track record of mentoring new investigators. He is, and will remain, a closely-involved mentor, committed to the success of Dr. Afkarian's training, her proposed studies and career development. Research: Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. However, with the current standards of care, we can only delay progression of this disease. This is partly due to a lack of effective therapies and partly due to absence of reliable assays for diagnosis of the early disease, before renal damage is severe or irreversible. New diagnostic and therapeutic tools are urgently needed, both of which require an improved understanding of the disease natural history, i.e. identification of new markers of onset and progression. Comparative proteomic analysis of urine (study of the urine protein composition and how it is changed in the disease state) has been used to identify markers for several kidney diseases. Dr. Thadhani (mentor)'s group has previously used this approach to identify a peptide signature that detected signs of early diabetic kidney injury up to 10 years prior to clinical diagnosis. This proposal lays out a multi-stage approach to identify the proteins that are altered in early DN. The preliminary data describes calibration and adaptation of a sensitive proteomic technology (iTRAQ: isobaric Tags for Relative and Absolute Quantitation) for application to urine. In Aim 1A, iTRAQ will be used to compare urine samples from patients with and without DN (cases and controls, respectively). This hypothesis-free and unbiased comparative analysis will generate a preliminary list of candidate biomarkers for early DN. In Aim 1B, this list will be supplemented with hypothesis-driven candidates, i.e. those with significant evidence of involvement in DN. Examples are proteins which (a) are part of pathways incriminated in DN, (b) have altered mRNA expression in DN or (c) have altered protein level in DN. This step serves to incorporate all currently available information into this biomarker discovery effort. In Aim 3, a target-specific and quantitative assay, e.g. ELISA, is used to re-evaluate the expression of putative biomarkers from Aims 1A and 1B in an independent cohort. This step will help weed out the false positives and identify a subset of markers whose differential expression in DN is confirmed. In Aim 4, the expression of these confirmed markers will be characterized over the course of disease. This proposal is designed to generate a group of candidate biomarkers for early DN which have been validated within the original study population. A future step would be to test these candidates in a broader and more varied population. In long term, we hope that the validated biomarkers would serve as a novel diagnostic tool for early DN and also help shed light on the disease pathophysiology. PUBLIC HEALTH RELEVANCE: Diabetes is the leading cause of end-stage kidney disease and dialysis in the US. The current medical tests can diagnose the disease only after significant and sometimes irreversible damage has already occurred. We plan to find urine marker proteins that can be used to detect early signs of diabetic damage to the kidneys, when it is more likely to be stopped or reversed.