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  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

摘要

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

引言

The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function1-4.

GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron5. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine.

The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.

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研究方案

在此之前任何动物的过程中，机构的动物护理和使用委员会（IACUC）必须批准该协议。该协议被批准为密歇根州立大学IACUC。

FITC的 - 菊粉解决方法1.实验前的准备

  1. 暖20毫升盐水至70℃，并慢慢地搅拌在100毫克的FITC-菊粉（5毫克/ ml的FITC-菊粉），直到所有菊粉溶解。
  2. 凉溶液至室温，并添加了牛血清白蛋白800毫克（40毫克/毫升BSA，冻干粉末，基本上球蛋白自由，低内毒素，≥98％纯度通过琼脂糖凝胶电泳）。
  3. 过滤用滤纸（1级）的菊粉BSA解决方案。放置在20ml的注射器用注射器尖过滤器（0.2微米）过滤溶液，并盖上铝箔避光。

2，麻醉和手术

  1. 将大鼠在感应腔室填充有5％异氟烷诱导麻醉。记录BODŸ重量（250-350克），将老鼠的目的是保持整个实验37℃的体温加热的手术平台。轻轻固定鼠与实验室磁带在爪子的平台。维持麻醉用1-2％异氟醚与医疗级100％O 2在0.8-1.0升/分钟的气流速率。
  2. 插入一锥形导管（血管内尖端外径，2.7F）插入股动脉血压和心脏速率监控和血液取样。
  3. 插入一个导管（PE-50）插入股静脉菊糖输注。固定导管周围组织与5-O丝编织手术缝合6。
  4. 附加的动脉导管以应变仪压力传感器。记录血压，并使用数据采集软件和显示在实时计算机屏幕上心脏速率。该技术表现出在细节上视频6。
  5. 经耻骨上切口暴露膀胱。切一小孔在膀胱的前端与插入套管（PE-190）用热扩口顶端的膀胱尿液收集内部。固定套管用一个荷包缝合膀胱。

3，尿液和血液采集

  1. 放置的FITC-菊粉的注射器在注射器泵的流速设置每100g体重1毫升/小时（3毫升/小时的大鼠称重300克）。附加注射器向股静脉导管。启动菊粉输液，并允许1-2小时平衡期。保持注射器用铝箔纸覆盖避光。
  2. 确定尿的流速是稳定和足够的试样分析（20微升/分钟）通过收集在预称重的收集瓶尿样，为期10分钟。重力测定尿量用数字刻度。足够的尿量为10分钟收集期为0.2毫升继续收集尿液样本，直到连续两个集合指示20微升/ m的尿流率在以上。
  3. 预药物样本
    1. 收集在一个20分钟期间的尿液样本。收集血液样品（0.5毫升），从动脉导管在尿液收集期间的中点。小心生理盐水彻底清除动脉导管含1 U肝素收集瓶采集血样前。使用收集瓶与体积标记，以促进为0.5ml的动脉血收集。
    2. 冲洗动脉导管与肝素盐水（20单位/毫升）以清除血液（约0.1ml）中的导管。动脉导管的长度应尽可能地短，以限制冲洗所需肝素盐水的体积。
      注:稀释血液样本产生GFR和Na和K的排泄分数的计算不准确
    3. 等待10分钟，并重复一第二预药物尿液和血液样品的集合。
  4. 下面的两个预毒​​品样品的收集，管理利尿DRU克，速尿（10毫克/千克），通过动脉导管。冲洗动脉导管与肝素生理盐水以清除药物的导管。照顾，以防止空气注入通过动脉导管。记录速尿注射的时间。
  5. 用药后的样品:在每个以下3个时间点的，收集在10分钟收集时段尿样和血样（0.5毫升）的尿液收集期间的中点。
    1. 对于用药后的样品1 - 收集5分钟后，速尿。
    2. 对于用药后的样品2 - 收集10分钟后，速尿。
    3. 对于用药后的样品3 - 收集15分钟后，速尿。
  6. 毕竟样品已收集，安乐死大鼠按照制度程序通过开胸手术和摘取心脏。取出两个肾脏​​。解封装（去掉周围的膜），并吸干肾脏，去除多余血液。权衡肾脏。

4。样品分析

  1. 衡量所有尿样体积与重量分析的数字称重，并记录重量。
  2. 离心的全血样品与台式离心机（1800×g离心）以分离血浆。转移血浆样本，以小标记的小瓶。
  3. 与钠/钾分析器分析Na和K浓度的尿和血浆样品中。
  4. 的FITC-菊粉的血浆和尿的测量
    1. 稀预药物尿（从1:200至1:400），以及后药物尿（1:10）与HEPES缓冲液（500毫米，pH值7.4）。
    2. 添加40微升HEPES缓冲标准品或样品和60微升的96孔板（每孔样品）中，并允许混合10分钟，同时用铝箔覆盖。
    3. 产生的标准曲线的FITC-菊粉的浓度6.25，12.5，25，50，100，200，400微克/毫升（ 图1）。确定在样品和标准的FITC-菊粉荧光用酶标仪与前分别为485和538纳米，引文和发射波长。
    4. 适合的标准荧光值到4放慢参数逻辑函数的回归分析。回归函数参数来计算血浆和尿液样品（ 见表1）中的FITC-菊粉浓度。

结果5后的实验室分析:计算

  1. 计算尿流量（UV;毫升/分钟）:尿体积收集（毫升）]÷[集合的时间（min）]
  2. 计算肾小球滤过率（GFR毫升/分钟）:尿菊糖浓度（微克/毫升）×紫外线（毫升/分钟）]÷[等离子体菊粉浓（微克/毫升）]
  3. 计算筛选钠负荷（微摩尔/分钟）:血浆钠浓度（微摩尔/毫升）×GFR（毫升/分钟）
  4. 计算钠排泄率（U 娜伏;微摩尔/分钟）:尿钠浓度（微摩尔/毫升）×UV（毫升/分钟）
  5. 计算分数排泄钠（FE娜;％）:[U 娜 V（微摩尔/分钟）]÷[滤过钠负荷（微摩尔/分钟）]×100
  6. 过滤计算负荷钾（微摩尔/分钟）:血钾浓度（微摩尔/毫升）×GFR（毫升/分钟）
  7. 计算钾排泄率（Uķ伏;微摩尔/分钟）:尿钾浓度（微摩尔/毫升）×UV（毫升/分钟）
  8. 计算分数排泄钾（FE K表;％）:[UķV（微摩尔/分钟）]÷[过滤钾负荷（微摩尔/分钟）]×100

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结果

在实验室演示中使用的利尿剂是呋塞米其非常迅速地抑制Na和K的重吸收过滤通过药物给药后几分钟内肾脏导致增加的Na，K和水排泄。由它的主要机制，速尿应该对GFR和Na和K的过滤载荷的影响最小，但会增加尿流Na和K，以及排泄分数

在表的代表性的结果3可知 ，在麻醉的大鼠，对于GFR预药物值的平均值为3.2毫升/分钟，钠排泄为0.58微摩尔/分钟（经过滤的...

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讨论

适当的标记物GFR测量必须符合四个标准:自由过滤在肾小球，可以绑定到血浆蛋白，并没有被吸收，也不分泌的肾单位。菊糖是一种果糖聚合物满足这些标准。其结果是，菊糖的肾清除被认为是金标准测定GFR 7。所表现出的技术代表确定菊粉8,9的恒定输注期间使用定时尿收集菊粉的肾清除率的传统方法。传统菊粉测量已用蒽酮法以产生由分光光度计10,11测得的比色定量测定?...

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披露声明

作者宣称，他们有没有竞争的财务权益。该意见或此处包含的断言是作者的个人观点，并不能解释为官方或反映了陆军部和国防部的意见。

致谢

资金来源为实验室示威是NIGMS授予:GM077119。我们感谢博士约瑟夫·海伍德和彼得·科贝特博士为他们的中西医结合和器官系统药理学支持短的:原因。我们也感谢汉娜·加弗女士的技术支持实验室演示。

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材料

NameCompanyCatalog NumberComments
5-0 Braided Silk Surgical SutureSurgical Specialties CorpSP1033
Assay Plate, 96-WellCostar 3922
Bovine Serum AlbuminSigma Chemical CoA2934-25G
CentrifugeBeckman CoulterMicroFuge 18, 357160
Conical Sample TubesDot Scientific Inc. #711-FTG
Cotton Tipped ApplicatorsSolon Manufacturing Co56200
Data Acquisition SoftwareADInstrumentsLabChart Pro 7.0
Digital Scale Denver InstrumentAPX-4001
FITC-InulinSigma Chemical CoF3272-1G
Gauze SpongesCovidien2146
Heated Surgical BedEZ-AnesthesiaEZ-212
HeparinSagnetNDC 25021-402-10
HEPESSigma Chemical CoH3375
IsofluraneAbbott Animal HealthIsoFlo, 5260-04-05
Isoflurane VaporizerEZ-AnesthesiaEZ-190F
Micro Dissecting ForcepsBiomedical Research Instruments Inc.70-1020
Microplate Reader - FluoroskanThermoScientificAscent FL, 5210460
NOVA 5+ Sodium/Potassium AnalyzerNOVA BioMedical14156
Olsen-Hegar Needle Holders with ScissorsFine Science Tools12002-12
PE-190 (for bladder catheter)BD Medical427435
Pressure Transducer ADInstrumentsMLT1199
Pyrex Culture TubesCorning Inc.99445-12
Rat Femoral Tapered Artery CatheterStrategic Applications Inc.RFA-01
Salix Furosemide 5%Intervet#34-478
Strabismus ScissorsFine Science Tools14075-11
Student Surgical ScissorsFine Science Tools91402-12
Surgical GlovesKimberly-ClarkSterling Nitrile Gloves
Syringe pumpRazel ScientificR99-E
Tissue ForcepsFine Science Tools91121-12
Tissue ScissorsGeorge Tiemann  Co105-420

5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar  3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.  #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale  Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader - Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer  ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann  Co 105-420

参考文献

  1. Silverthorn, D. U. Human Physiology: An integrated approach. , Pearson. (2012).
  2. Hall, J. E. Guyton and Hall Textbook of Medical Physiology. , 303-344 (2011).
  3. Levey, A. S. Measurement of renal function in chronic renal disease. Kidney International. 38 (1), 167-184 (1990).
  4. Thurau, K., Valtin, H., Schnermann, J. Kidney. Annual Review of Physiology. 30, 441-524 (1968).
  5. Shannon, J. A., Smith, H. W. The excretion of inulin, xylose, and urea by normal and phoriziniaed man. Journal of Clinical Investigation. 14, 393-401 (1935).
  6. Jespersen, B., Knupp, L., Northcott, C. A. Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements. Journal of Visualized Experiments. (59), (2012).
  7. Sterner, G., et al. Determining 'true' glomerular filtration rate in healthy adults using infusion of inulin and comparing it with values obtained using other clearance techniques or predictive equations. Scandinavian Journal of Urology and Nephrology. 42, 278-285 (2008).
  8. Toto, R. D. Conventional measurement of renal function utilizing serum creatinine, creatinine clearance, inulin and para-aminohippuric acid clearance. Current Opinion in Nephrology and Hypertension. 4 (6), 505-509 (1995).
  9. Matavelli, L. C., Kadowitz, P. J., Navar, L. G., Majid, D. S. Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats. Americam Journal of Physiology. 296, F170-F176 (2009).
  10. Davidson, W. D., Sackner, M. A. Simplification of the anthrone method for the determination of inulin in clearance studies. Journal of Laboratory, & Clinical Medicine. 62, 351-356 (1963).
  11. Symes, A. L., Gault, M. H. Assay of inulin in tissues using anthrone. Clinical Biochemistry. 8 (1), 67-70 (1975).
  12. Shalmi, M., Lunau, H. E., Petersen, J. S., Bak, M., Christensen, S. Suitability of tritiated inulin for determination of glomerular filtration rate. Americam Journal of Physiology. 260 (2 Pt 2), F283-F289 (1991).
  13. Denton, K. M., Anderson, W. P. Glomerular untrafiltration in rabbits with superficial glomeruli. EUropean Journal of Physiology. 419 (3-4), 235-242 (1991).
  14. Jobin, J., Bonjour, J. -P. Measurement of glomerular filtration rate in conscious unrestrained rats with inulin infused by implanted osmotic pumps. Americam Journal of Physiology. 248 (5 Pt 2), F734-F738 (1985).
  15. Lorenz, J. N., Gruenstein, E. A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin. Americam Journal of Physiology. 276 (1 Pt 2), F172-F177 (1999).
  16. Qi, Z., et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Americam Journal of Physiology. 286 (3), F590-F596 (2004).
  17. Bivona, B. J., Park, S., Harrison-Bernard, L. M. Glomerular filtration rate determinations in conscious type II diabetic mice. Americam Journal of Physiology. 300 (3), F618-F625 (2011).
  18. Rosen, S. M. Effects of anaesthesia and surgery on renal hemodynamics. British Journal of Anesthesiology. 44, 252-258 (1972).
  19. Cousins, M. J. Anesthesia and the kidney. Anaesthesia and intensive care. 11 (4), 292-320 (1983).
  20. Walter, S. J., Zewde, T., Shirley, D. G. The effect of anaesthesia and standard clearance procedures on renal function in the rat. Quarterly Journal of Experimental Physiology. 74, 805-812 (1989).
  21. Rieg, T. A. A high-throughput method for measurement of glomerular filtration rate in conscious mice. Journal of Visualized Experiments. (75), (2013).

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