在肾脏皮质提取物中演示血管内皮生长因子与叶黄素激素之间的线性关系
Summary
这里介绍的是利用皮质肾提取物制备和总蛋白质正常化，以证明血管内皮生长因子和哺乳动物肾脏叶黄素激素之间的相关性的协议。
Abstract
血管内皮生长因子 （VEGF） 有助于控制肾脏的血管生成和血管渗透性。肾病，如糖尿病肾病，与肾脏的VEGF调节不良有关。肾脏生理条件下控制VEGF的因素没有很好地理解。叶黄激素 （LH）， 一种亲血管激素， 有助于调节生殖器官的生理 VEGF 表达.鉴于LH受体在肾脏中被发现，我们齐希克研究所在这里假设，LH也有助于调节肾脏中的VEGF表达。为了提供证据，我们旨在证明LH水平能够预测哺乳动物肾脏的VEGF水平。大多数涉及肾脏的VEGF相关调查都使用低阶哺乳动物作为模型（即啮齿动物和兔子）。为了将这项工作转化为人体，决定以高阶哺乳动物（即牛和猪模型）检查VEGF和LH之间的关系。该协议使用来自肾脏皮层的总蛋白质莱沙。这种方法成功的关键包括：死后立即从屠宰场动物那里采购肾脏，以及通过总蛋白质使麻醉剂水平（在肾脏提取物中）正常化。本研究成功地证明了牛和猪肾中的LH和VEGF之间的显著线性关系。结果在两个不同的物种中可重现。该研究提供了支持证据，证明使用牛和猪的肾脏提取物是肾脏生理学研究的优良、经济、丰富的资源，特别是用于检查VEGF与其他分析物之间的相关性。
Introduction
血管内皮生长因子A（VEGF-A），有助于调节血管生成和血管渗透性在肾脏和其他器官1，2（下称VEGF-A将称为VEGF）。肾脏的VEGF水平处于严格的静时控制之下。当肾VEGF水平升高或抑郁时，肾脏可能出现故障。例如，在出生后3周内，患有VEGF特异性杂合性小鼠会发展成内皮病和无血球蛋白（即，在人类先兆子宫颈癌中看到的肾病变），到3个月大时，这些异体细胞发生末期肾衰竭。波多细胞特异性同源性敲除在出生3，4的1天内死于水肿和肾衰竭。
另一方面，肾VEGF的过度表达导致蛋白尿和肾小球肥大3，4。例如，过度表达VEGF的转基因兔子在肾病早期表现出渐进蛋白尿，肾病早期肾球菌过滤率增加，随后在后期下降的球状过滤率3。糖尿病肾病是糖尿病成人终末期肾病的主要原因，与VEGF调节不良密切相关。在病理条件下，缺氧在诱导VEGF表达中的作用受到很大重视。然而，在生理条件下（肾脏和其他器官）控制VEGF的因素并不十分清楚2，6。识别与生理和病理VEGF调节有关的这些因素（氧气除外）是一项重要工作。
叶黄激素（LH），一种亲血管激素，有助于调节生殖器官（如卵巢和睾丸7、8）的生理VEGF表达。先前的研究已经提供了证据，LH也有助于调节非生殖器官的VEGF，如眼睛6，9，10。LH受体在肾的髓叶和皮层发现11，12。值得注意的是，肾管上皮细胞，以及LH受体，表达VEGF11，12，13，14。结合这两个观察，我们假设LH也有助于调节肾中的VEGF表达13，14。为了提供这种LH/VEGF关系的证据，提出的协议旨在表明LH水平能够预测肾脏中的VEGF水平。以前许多涉及肾脏的VEGF相关调查都使用了低阶哺乳动物模型（即啮齿动物和兔子）2。为了将这项工作转化为人体，该研究在高阶哺乳动物（这里，牛和猪模型）中考察了VEGF和LH之间的关系。为了达到这个目标，从牛和猪肾的皮层区域制备了总蛋白莱沙。
Protocol
Representative Results
Discussion
动物死亡后立即从屠宰场获取肾脏是这种方法成功的关键。这是利用牛和猪器官而不是人类尸体的主要优势。通常从死亡时间到人类尸体器官被采购至少需要12-24小时。由于人体器官的化学成分在15日2小时内发生显著变化，16日，人类尸体肾脏的VEGF研究可能并不反映真实情况。尽管该议定书非常强调在提取动物器官后立即采购和放置在冰上的重要性，但…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢肖尔的屠宰场（布利斯菲尔德，MI）提供牛和猪肾。这项研究没有利用赠款资金。
Materials
| Bovine LH ELISA Kit | MyBiosource, San Diego, CA. | MBS700951 | |
| Bovine VEGF-A ELISA Kit | MyBiosource, San Diego, CA. | MBS2887434 | |
| Micro BCA Protein Assay Kit | ThermoFisher Scientific Inc, Columbus, OH. | 23235 | |
| Porcine LH ELISA Kit | MyBiosource, San Diego, CA. | MBS009739 | |
| Porcine VEGF-A ELISA | Ray Biotech, Norcross, GA. | ELP-VEGFA-1 | |
| RIPA Lysis and Extraction Buffer | ThermoFisher Scientific Inc, Columbus, OH. | 89901 |
References
- Advani, A., et al. Role of VEGF in maintaining renal structure and function under normotensive and hypertensive conditions. Proceedings of the National Academy of Science U. S. A. 104 (36), 14448-14453 (2007).
- Majumder, S., Advani, A. VEGF and the diabetic kidney: More than too much of a good thing. Journal of Diabetes and its Complications. 31 (1), 273-279 (2017).
- Liu, E., et al. Increased expression of vascular endothelial growth factor in kidney leads to progressive impairment of glomerular functions. Journal of the American Society of Nephrology. 18 (7), 2094-2104 (2007).
- Eremina, V., et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. Journal of Clinical Investigation. 111 (5), 707-716 (2003).
- Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocrine Reviews. 25 (4), 581-611 (2004).
- Movsas, T. Z., Sigler, R., Muthusamy, A. Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes. Current Eye Research. 43 (8), 1041-1044 (2018).
- Babitha, V., et al. Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells. General and Comparative Endocrinology. 198, 1-12 (2014).
- Trau, H. A., Davis, J. S., Duffy, D. M. Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2. Biology of Reproduction. 92 (1), 15 (2015).
- Movsas, T. Z., et al. Confirmation of Luteinizing Hormone (LH) in Living Human Vitreous and the Effect of LH Receptor Reduction on Murine Electroretinogram. Neurosciences. 385, 1-10 (2018).
- Movsas, T. Z., Sigler, R., Muthusamy, A. Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development. Current Eye Research. 43 (10), 1286-1289 (2018).
- Hipkin, R. W., Sanchez-Yague, J., Ascoli, M. Identification and characterization of a luteinizing hormone/chorionic gonadotropin (LH/CG) receptor precursor in a human kidney cell line stably transfected with the rat luteal LH/CG receptor complementary DNA. Molecular Endocrinology. 6 (12), 2210-2218 (1992).
- Lei, Z. M., et al. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Molecular Endocrinology. 15 (1), 184-200 (2001).
- Schrijvers, B. F., Flyvbjerg, A., De Vriese, A. S. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney International. 65 (6), 2003-2017 (2004).
- Apaja, P. M., Aatsinki, J. T., Rajaniemim, H. J., Petaja-Repo, U. E. Expression of the mature luteinizing hormone receptor in rodent urogenital and adrenal tissues is developmentally regulated at a posttranslational level. Endocrinology. 146 (8), 3224-3232 (2005).
- Ondruschka, B., et al. Post-mortem in situ stability of serum markers of cerebral damage and acute phase response. International Journal of Legal Medicine. 133 (3), 871-881 (2019).
- Swain, R., et al. Estimation of post-mortem interval: A comparison between cerebrospinal fluid and vitreous humour chemistry. Journal of Forensic and Legal Medicine. 36, 144-148 (2015).
- Thompson, C. S., Traynor, I. M., Fodey, T. L., Faulkner, D. V., Crooks, S. R. H. Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor. Talanta. 172, 120-125 (2017).
- Konstantinou, G. N. Enzyme-Linked Immunosorbent Assay (ELISA). Methods in Molecular Biology. 1592, 79-94 (2017).
- Levesque, B. M., et al. Low urine vascular endothelial growth factor levels are associated with mechanical ventilation, bronchopulmonary dysplasia and retinopathy of prematurity. Neonatology. 104 (1), 56-64 (2013).
- Leviton, A., et al. Antecedents and early correlates of high and low concentrations of angiogenic proteins in extremely preterm newborns. Clinica Chimica Acta. 471, 1-5 (2017).
- Simo-Servat, O., Hernandez, C., Simo, R. Usefulness of the vitreous fluid analysis in the translational research of diabetic retinopathy. Mediators of Inflammation. , 872978 (2012).
- Sharma, R. K., Rowe-Rendleman, C. L. Validation of molecular and genomic biomarkers of retinal drug efficacy: use of ocular fluid sampling to evaluate VEGF. Neurochemical Research. 36 (4), 655-667 (2011).
