Name: estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis
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作者感谢所有研究参与者和 UAB CCTS 生物营养核心和 UAB 高分辨率成像服务中心的贡献。这项工作得到了国家卫生研究院DK106284和DK123542（TM）和UL1TR003096（国家推进转化科学中心）的支持。

这项工作中概述的所有实验都得到了阿拉巴马大学伯明翰分校（UAB）机构审查委员会的批准。健康成人（33.6±3.3岁;n=10）如果有正常的血液综合代谢板，非烟草使用者，非怀孕，体重指数在20-30公斤/米2之间，并且没有慢性疾病或急性疾病，则报名参加研究。健康参与者在研究开始前签署了书面知情同意书。
1. 临床协议和尿液收集
<ol><li>让参与者在收集尿液（24 小时?...

<ol>
	<li>Fogazzi, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystalluria:+a+neglected+aspect+of+urinary+sediment+analysis.">Crystalluria: a neglected aspect of urinary sediment analysis.</a> Nephrology, Dialysis, Transplantation. 11 (2), 379-387 (1996).</li><li>Kuo, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urine+calcium+and+volume+predict+coverage+of+renal+papilla+by+Randall's+plaque.">Urine calcium and volume predict coverage of renal papilla by Randall's plaque.</a> Kidney International. 64 (6), 2150-2154 (2003).</li><li>Robertson, W. G., Peacock, M., Nordin, B. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracrystalline+and+surface-bound+proteins+on+the+attachment+of+calcium+oxalate+monohydrate+crystals+to+renal+cells+in+undiluted+human+urine.">The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine.</a> BJU International. 105, 708-715 (2010).</li><li>Bader, C. A., Chevalier, A., Hennequin, C., Jungers, P., Daudon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+aspects+of+spontaneous+crystalluria+studies+in+calcium+stone+formers.">Methodological aspects of spontaneous crystalluria studies in calcium stone formers.</a> Scanning Microscopy. 8 (2), 215-231 (1994).</li><li>Daudon, M., Cohen-Solal, F., Jungers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eurolithiasis. 9th European Symposium on Urolithiasis. , 261-263 (2001).</li><li>Werness, P. G., Bergert, J. H., Smith, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystalluria.">Crystalluria.</a> Journal of Crystal Growth. 53 (1), 166-181 (1981).</li><li>Fan, J., Chandhoke, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+crystalluria+in+freshly+voided+urines+of+recurrent+calcium+stone+formers+and+normal+individuals+using+a+new+filter+technique.">Examination of crystalluria in freshly voided urines of recurrent calcium stone formers and normal individuals using a new filter technique.</a> Journal of Urology. 161 (5), 1685-1688 (1999).</li><li>Sun, X. Y., Ouyang, J. M., Yu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-dependent+cellular+toxicity+on+renal+epithelial+cells+and+stone+risk+of+calcium+oxalate+dihydrate+crystals.">Shape-dependent cellular toxicity on renal epithelial cells and stone risk of calcium oxalate dihydrate crystals.</a> Scientific Reports. 7 (1), 7250 (2017).</li><li>He, J. Y., Deng, S. P., Ouyang, J. 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NTA在本研究中使用钙结合探针Flo-4 AM来评估人类尿液中的纳米晶体。没有标准方法可以检测尿液中的纳米晶体。一些研究小组已经检测出尿液中的纳米晶体，并依靠使用广泛的协议或方法，这些协议或方法限制了他们量化样本的能力。这项研究展示了一种特定而敏感的方法，用于检测参与饮食喂养研究的人类尿液中含有纳米晶体的钙，该研究包括摄入高...

当尿液中富含矿物质时，尿晶体就会形成。这可能发生在健康的人，但更常见的个人肾结石1。泌尿晶体的存在和积累会增加患肾结石的风险。具体来说，当晶体与兰德尔的斑块结合，核化，积累，并随着时间的推移生长2，3，4时，就会发生这种情况。晶体在肾结石形成之前和晶体的评估可能具有预测价值的肾结石前3，5。具体来说，结晶体已被建议有用，以预测在含有结石6，7的草酸钙病史患者的结石复发的风险。
据报道，晶体对肾上皮和循环免疫细胞功能8、9、10、11、12、13产生负面影响。此前有报道称，与健康个体相比，从草酸钙（CaOx）肾结石前体中循环的单核细胞抑制了细胞生物能学。此外，CaOx晶体可减少细胞生物能学，并破坏单核细胞8中的红氧平衡。食用富含草酸盐的膳食可能导致结晶体，导致肾管损伤，并改变保护肾结石形成的尿大分子的生产和功能。多项研究已证明，尿晶体的形状和大小可能因尿液17、18、19的pH值和温度而异。此外，尿蛋白已被证明可以调节晶体行为20。道登等人19日提出，结晶体分析可能有助于肾结石病患者的管理，并评估他们对治疗的反应。目前可用于评估晶体存在的一些常规方法包括极化显微镜21、22、电子显微镜23、粒子计数器3、尿液过滤24、蒸发3、5或离心21。这些研究为肾结石领域提供了宝贵的见解。然而，这些方法的局限性是无法可视化和量化大小小于 1μm 的晶体。这种大小的晶体可能通过附着在兰德尔的斑块上来影响CaOx石的生长。
与较大的微晶体25相比，纳米晶体已证明对肾脏细胞造成广泛损伤。纳米晶体的存在已经报告在尿液中使用纳米粒子分析仪26，27。最近的研究已经使用荧光标记的双磷酸盐探针（亚龙-氟辛/阿伦德龙酸盐-Cy5）来检查纳米晶体使用纳米级流细胞测量28。这种染料的限制是，它不是具体的，将结合几乎所有类型的石头，除了半胱氨酸。因此，准确评估个体中纳米晶体的存在可能是诊断结晶体和/或预测结石风险的有效工具。本研究的目的是利用纳米粒子跟踪分析（NTA）检测和量化含有纳米晶体的钙（大小为 <1微米）。为此，NTA 技术与含钙氟磷、氟-4 AM 相结合，用于检测和量化健康成年人尿液中含有纳米晶体的钙。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Jouan Centrifuge</td>
			<td>CR3-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Oxalate monohydrate</td>
			<td>Synthesized in the lab as previously described29.</td>
			<td> </td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Calcium Phosphate crystals (hydroxyapatite nanopowder)</td>
			<td>Sigma</td>
			<td>677418</td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fischer Scientific</td>
			<td>AC615095000</td>
			<td>Store at RT; Stock 100%</td>
		</tr>
		<tr>
			<td>Fluo-4 AM*</td>
			<td>AAT Bioquest, Inc.</td>
			<td>20550</td>
			<td>Store at Freezer (-20&#176;C); Stock 5 mM</td>
		</tr>
		<tr>
			<td>Gold Nanoparticles</td>
			<td>Sigma</td>
			<td>742031</td>
			<td>Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>NanoSight Instrument</td>
			<td>Malvern Instruments, UK</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>98-4730</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon Disinfectant</td>
			<td>LanXESS Energizing Company, Germany</td>
			<td>LSP</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20&#176;C.</td>
		</tr>
	</tbody>
</table>

estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis

肾结石在全世界成人和儿童中越来越普遍。最常见的肾结石类型由草酸钙（CaOx）晶体组成。当尿液中富含矿物质（如钙、草酸盐、磷酸盐）并形成肾结石之前时，就会发生结晶。评估石器晶体的标准方法包括显微镜、过滤和离心。然而，这些方法主要检测微晶体，而不是纳米晶体。纳米晶体被建议对肾脏上皮细胞的危害大于体外微晶体。在这里，我们描述了纳米粒子跟踪分析（NTA）检测人类泌尿纳米晶体的能力。健康成年人在饮用草酸盐负荷以刺激泌尿纳米晶体之前，先吃受控的草酸盐饮食。尿在草酸盐负荷前后收集了24小时。用乙醇加工和清洗样品以净化样品。泌尿纳米晶体沾染了钙结合氟磷，氟-4 AM。染色后，使用 NTA 确定纳米晶体的大小和计数。这项研究的结果表明，NTA可以有效地检测健康成年人的纳米晶体。这些发现表明，NTA可能是肾结石患者纳米晶体的有价值的早期检测方法。

这项研究的结果表明，NTA可以有效地检测人类尿液中含有尿纳米晶体的钙的平均大小和浓度。这是通过使用氟磷、氟-4 AM 和纳米粒子跟踪分析实现的。Fluo-4 AM 能够与 CaOx 和 CaP 晶体结合。如图3A所示，CaOx晶体的大小确定在50-270纳米之间，平均浓度为1.26 x 109颗粒/mL。CaP晶体的大小在30-225纳米之间，平均浓度为2.22×109粒子/mL（图3B）。为了...

Watch this Scientific Journal Video about Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis at JoVE.com

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

这项研究的目的是确定纳米粒子跟踪分析（NTA）能否检测和量化含有健康成年人纳米晶体的尿钙。目前的研究结果表明，NTA可能是估计肾结石疾病期间泌尿纳米晶体的潜在工具。

使用氟磷钙标签和纳米粒子跟踪分析估计人类泌尿纳米晶体

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

本协议可以专门检测人类尿液中含钙的纳米晶体。这项技术可能有助于预测肾结石形成的早期阶段以及与晶体尿素相关的其他疾病。证明这个程序的将是帕文·库马尔博士，我实验室的研究员IV。让参与者在三天内食用低草酸盐饮食，并在一夜之间快速食用。然后从参与者收集24小时的尿液样本，然后让他们消耗草酸盐。给参与者喂一个含有水果和蔬菜的冰沙，草酸盐负荷约为8毫摩尔。让参与者在牛皮后24小时收集尿液，并在第二天返回。在处理前在室温下保持所有尿液样本。测量和记录尿液 pH 和尿样体积。彻底混合，将 50 毫升尿液加入标记的无菌 50 毫升圆锥管中。使用台式离心机在室温下将样品在 1200 倍 G 下离心 10 分钟。丢弃超自然物，然后用五毫升100%乙醇再次清洗并补充颗粒。使用台式离心机在室温下将样品在 1200 倍 G 下离心 10 分钟。丢弃超自然物，在100%乙醇的一毫升中补充颗粒。将样品存放在零下20摄氏度以供以后处理，或立即对样品进行染色。以1：1000的比例在超纯水中稀释100纳米大小的金纳米粒子，并用于优化仪器的设置。在用5毫升Flo-4 AM染色之前，在水中稀释尿液样本20次，在黑暗中30分钟，使用NTA分析样本。在 NTA 分析之前，准备文本稿件中描述的草酸钙和磷酸钙晶体。打开计算机和仪器，然后打开软件并打开相机。单击窗口左上角的捕获图标以启动捕获模式。首先使用一毫升注射器将空气泵入平台，直到平台看起来清洁。轻轻地将水加入仪器两到三次，并使用另一个一毫升注射器去除任何气泡。平台清洁后，加入水，通过查看摄像机检查表面是否有污染，然后将金纳米粒子作为控制样品装载泵喷油器，以设置仪器。将屏幕上或旋钮上的摄像头级别调整到仪器的右侧，直到图像开始显示彩色像素，然后降低相机水平。接下来，调整屏幕以优化图像。左键单击视频图像上的鼠标按钮。按住左鼠标按钮，上下拖动图像以获取整个视图。设置输液速度并对焦相机，使金纳米粒子在相机屏幕上可见。将输液速度设置为高，以便进行初始设置，以确保检测到金纳米粒子。一旦检测到，将速度降低到每分钟 50 微升，以可视化金纳米粒子。调整相机水平以可视化粒子。对于未染色的样品，在五级调整屏幕增益，以实现相机对焦，并将相机级别设置为 8。设置焦点后，记录示例。优化后，在评估其他样品之前，再次用水清洁设备，以确保管子清洁且颗粒不存在。要分析染色样品，将相机调整到包含适当荧光滤光片的滤光片位置，并将稀释和染色样品加载到样品装载泵喷油器上，将速度降低到每分钟 20 微升，以便进行分析。将屏幕增益调整为 5，将摄像机级别调整为 13。每次测量后保存数据。计算每个样本所有五个读数的纳米粒子的平均数，并使用平均值的标准偏差或标准误差分析数据。使用 T 测试进行配对分析。Fluo-4 AM 能够与草酸钙晶体结合，这些晶体的大小在 50 到 270 纳米之间，平均浓度为每毫升 12.6 亿颗粒，磷酸钙晶体的平均浓度在 30 至 225 纳米之间，平均浓度为每毫升 22.2 亿颗粒。在草酸盐负荷之前收集的24小时尿液样本中含有一些大小在110至300纳米之间的尿纳米晶体。相比之下，后氧化物样品中的泌尿纳米晶体显著增加。为了确认该方法的可重复性，对样品进行了三次测量，技术复制品没有显著变化。在尝试此协议时，在机器中添加没有气泡的样品可能会遇到一些困难。为了防止这种情况，在机器中加入空气和水，然后慢慢添加样品。此外，请务必持续监控样品，并且不存在气泡。这种新方法将在患有草酸钙肾结石病的个体中测试，以监测疾病进展和/或预测肾结石风险。

Research

JoVE Journal

Medicine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI for Cell Tracking

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

诱导和人类劳力骨骼肌损伤评估

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

使用 PubMed、Scopus 和科学数据库网络比较书目分析

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

酸化浸渍法评价全肾肾素数

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...