Hepcidin Lateral Flow Device for Prediction of Acute Kidney Injury

描述(申请人提供)：急性肾损伤(AKI)是体外循环(CPB)辅助手术中常见的严重并发症，包括冠状动脉旁路移植、瓣膜置换、主动脉瘤和器官移植。老年患者、糖尿病患者和慢性肾脏疾病(CKD)患者术后发生AKI的风险非常高。每年有100万例CPB手术导致数千例与AKI相关的死亡和残疾，涉及肾脏替代疗法(RRT)，累计花费数十亿美元。现有的确认AKI患者功能丧失的肾脏标志物(如血肌酐)是诊断AKI的很晚的标志物(1-3天)。很少有生物标记物(如NGAL、KIM-1)可用于AKI的早期预测。预测CPB术后AKI严重程度的早期生物标志物和快速测试将有助于患者分层、早期治疗干预和药物开发。最近，我们发现海普西丁是一种在肝脏产生的小肽(2.78kD)，在尿液中排泄，可能是CPB辅助手术后AKI的良好生物标志物。在与我们在澳大利亚的合作者进行的两项100名患者的临床试验中(NCT00910221；Prowle等人。2012)和德国(NCT00672334；Haase-Felietz等人2011年)，分别在基线、基线后6小时和24小时采集血浆和尿样。与已建立的AKI生物标志物--血肌酐(PCr)和中性粒细胞明胶酶相关脂蛋白(NGAL)相反，在这两项研究中，当根据RIFLE标准(基于PCR)进行评分时，尿海普西汀(uHep；ng/ml)和调整尿肌酐的uHep(uHep/Ucr；ng/mg)与AKI的严重程度呈负相关。无AKI的患者在6小时内uHep和uHep/Ucr水平显著升高，并在24小时后升高。在德国的这项研究中，在6小时内，uHEP和uHep/Ucr分别是急性心肌梗死患者的3-7倍(P=0.004，P=0.002)，预测AUC-ROC0.80；0.88，预测AUC-ROC0.81；0.88，预测AUC-ROC0.81；0.88，预测AUC-ROC0.81；0.88。这些数据支持uHep和uHep/Ucr作为6小时内无AKI恢复的有前景的生物标志物，比现有的生物标志物如血肌酐、尿量和肾小球滤过率早得多。然而，为了使生物标记物对AKI的诊断有用，必须对其进行测量，并将数据迅速返回给外科医生和ICU工作人员，因为对AKI几乎没有既定的临床干预措施。我们建议开发一种便携式定量侧向流动装置(LFD)，该装置可以快速、定量地检测ICU中的尿液和血浆中的海普西丁浓度。我们最近在正在进行的SBIR研究中发现并鉴定了两种新的小鼠抗海普西丁单抗(MAb)(PA 08-114；2-R44-DK083843-02)。在我们的上一次提交中，我们使用了一种金标记的海普西丁示踪剂制作了一个原型海普西丁H1单抗LFD，但我们最终放弃了它。我们修订的第一阶段研究和开发工作的具体目标是：1)。评估来自前瞻性临床研究的1800份未确认的时间匹配样本中的尿庚西丁、NGAL和Kim-1，并进行广泛的统计分析；2)开发一种适用于血浆和尿庚西丁的快速、定量侧向流动装置(LFD)，并在前瞻性研究的临床样本中测试该新装置，以评估EPCIDin LFD在CPB后AKI中的应用。

DESCRIPTION (provided by applicant): Acute kidney injury (AKI) is a common, severe complication of cardiopulmonary bypass (CPB) assisted surgeries, and include coronary artery bypass grafts, valve replacements, aortic aneurisms, and organ transplants. Elderly patients, diabetics, and chronic kidney disease (CKD) patients, have very high risk of developing AKI postoperatively. One million CPB surgeries yearly result in thousands of AKI-related deaths and disabilities involving renal replacement therapy (RRT), cumulatively costing billions. Existing renal markers confirming loss of function (e.g. serum creatinine) in AKI are very late markers (1-3 days) for diagnosis of AKI. Few biomarkers (e.g. NGAL, KIM-1) are commercially available for early prediction of AKI. Early biomarkers and rapid tests predicting the severity of post-CPB AKI will enable patient stratification, earlier therapeutic interventions, and drug development for AKI Recently, we have shown that hepcidin is a small peptide (2.78kD) produced in the liver and excreted in urine and may be a good biomarker for AKI post CPB-assisted surgery. In two 100 patient clinical trials with our collaborators in Australia (NCT00910221; Prowle et al. 2012) and Germany (NCT00672334; Haase-Felietz et al. 2011), plasma and urine samples were taken at baseline, 6hr and 24hr from baseline. In contrast to established biomarkers of AKI, plasma creatinine (pCr) and neutrophil gelatinase-associated lipocalin (NGAL), in both these studies urinary hepcidin (uHep; ng/ml) and uHep adjusted for urine creatinine (uHep/uCr; ng/mg) was inversely correlated with severity of AKI when scored by RIFLE criteria (based on pCr). AKI-free patients had highly elevated levels of uHep and uHep/uCr at 6hr that increased through 24hr. uHep and uHep/uCr increased 3- to 7-fold compared to patients with AKI (P = 0.004, P = 0.002) at 6hr in the German study with a AUC-ROC 0.80; 0.88, respectively, for prediction of AKI-free recovery and an AUC-ROC 0.81; 0.88, respectively, for prediction of RRT-free recovery. These data support uHep and uHep/uCr as a promising biomarker of AKI-free recovery at 6 hours, much earlier than existing biomarkers such as plasma creatinine, urine output volume, and GFR. However, for a biomarker to be useful for diagnosis of AKI, it must be measured and the data returned to the surgeon and ICU staff quickly as there are few established clinical interventions for AKI. We propose development of a portable, quantitative lateral flow device (LFD) that can rapidly determine urine and plasma hepcidin concentrations rapidly and quantitatively in the ICU. We have recently discovered and characterized two new mouse anti-hepcidin monoclonal antibodies (MAb) in ongoing SBIR research (PA 08- 114; 2-R44-DK083843-02). For our last submission we made a prototype hepcidin H1 MAb LFD using a gold- conjugated hepcidin tracer that we ultimately abandoned. The specific aims for our revised Phase I research and development efforts are, 1). Assess urinary and plasma hepcidin, NGAL, and KIM-1 in 1800 de-identified time-matched samples from a prospective clinical study and perform extensive statistical analysis, and 2) Develop a rapid, quantitative lateral flow device (LFD) suitable for plasma and urine hepcidin and test the new device in the clinical samples from the prospective study to evaluate utility of a hepcidin LFD for post-CPB AKI.