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。

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<ol><li>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。 </li><li>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）。 </li><li>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。 </li></ol> 2，麻醉和手术<ol><li>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。 </li><li>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。 </li><li>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。 </li><li>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。 </li><li>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。 </li></ol> 3，尿液和血液采集<ol><li>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。 </li><li>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。 </li><li>预药物样本<ol><li>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。 </li><li>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</li><li>等待10分钟，并重复一第二预药物尿液和血液样品的集合。 </li></ol></li><li>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。 </li><li>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。 <ol><li>对于用药后的样品1 - 收集5分钟后，速尿。 </li><li>对于用药后的样品2 - 收集10分钟后，速尿。 </li><li>对于用药后的样品3 - 收集15分钟后，速尿。 </li></ol></li><li>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。 </li></ol><ol><li>衡量所有尿样体积与重量分析的数字称重，并记录重量。 </li><li>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。 </li><li>与钠/钾分析器分析Na和K浓度的尿和血浆样品中。 </li><li>的FITC-菊粉的血浆和尿的测量<ol><li>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）。 </li><li>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。 </li><li>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。 </li><li>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。 </li></ol></li></ol>结果5后的实验室分析:计算<ol><li>计算尿流量（UV;毫升/分钟）:尿体积收集（毫升）]÷[集合的时间（min）] </li><li>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）] </li><li>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） </li><li>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） </li><li>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 </li><li>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） </li><li>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） </li><li>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 </li></ol>

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</ol>

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。

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?...

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.

<table><tbody><tr><td>5-0 Braided Silk Surgical Suture</td><td>Surgical Specialties Corp</td><td>SP1033</td><td></td></tr><tr><td>Assay Plate, 96-Well</td><td>Costar&amp;nbsp;</td><td>3922</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Chemical Co</td><td>A2934-25G</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>MicroFuge 18, 357160</td><td></td></tr><tr><td>Conical Sample Tubes</td><td>Dot Scientific Inc.&amp;nbsp;</td><td>#711-FTG</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>Solon Manufacturing Co</td><td>56200</td><td></td></tr><tr><td>Data Acquisition Software</td><td>ADInstruments</td><td>LabChart Pro 7.0</td><td></td></tr><tr><td>Digital Scale&amp;nbsp;</td><td>Denver Instrument</td><td>APX-4001</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma Chemical Co</td><td>F3272-1G</td><td></td></tr><tr><td>Gauze Sponges</td><td>Covidien</td><td>2146</td><td></td></tr><tr><td>Heated Surgical Bed</td><td>EZ-Anesthesia</td><td>EZ-212</td><td></td></tr><tr><td>Heparin</td><td>Sagnet</td><td>NDC 25021-402-10</td><td></td></tr><tr><td>HEPES</td><td>Sigma Chemical Co</td><td>H3375</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott Animal Health</td><td>IsoFlo, 5260-04-05</td><td></td></tr><tr><td>Isoflurane Vaporizer</td><td>EZ-Anesthesia</td><td>EZ-190F</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Biomedical Research Instruments Inc.</td><td>70-1020</td><td></td></tr><tr><td>Microplate Reader - Fluoroskan</td><td>ThermoScientific</td><td>Ascent FL, 5210460</td><td></td></tr><tr><td>NOVA 5+ Sodium/Potassium Analyzer</td><td>NOVA BioMedical</td><td>14156</td><td></td></tr><tr><td>Olsen-Hegar Needle Holders with Scissors</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>PE-190 (for bladder catheter)</td><td>BD Medical</td><td>427435</td><td></td></tr><tr><td>Pressure Transducer&amp;nbsp;</td><td>ADInstruments</td><td>MLT1199</td><td></td></tr><tr><td>Pyrex Culture Tubes</td><td>Corning Inc.</td><td>99445-12</td><td></td></tr><tr><td>Rat Femoral Tapered Artery Catheter</td><td>Strategic Applications Inc.</td><td>RFA-01</td><td></td></tr><tr><td>Salix Furosemide 5%</td><td>Intervet</td><td>#34-478</td><td></td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools</td><td>14075-11</td><td></td></tr><tr><td>Student Surgical Scissors</td><td>Fine Science Tools</td><td>91402-12</td><td></td></tr><tr><td>Surgical Gloves</td><td>Kimberly-Clark</td><td>Sterling Nitrile Gloves</td><td></td></tr><tr><td>Syringe pump</td><td>Razel Scientific</td><td>R99-E</td><td></td></tr><tr><td>Tissue Forceps</td><td>Fine Science Tools</td><td>91121-12</td><td></td></tr><tr><td>Tissue Scissors</td><td>George Tiemann&amp;nbsp; Co</td><td>105-420</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p&gt;5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar&amp;nbsp; 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.&amp;nbsp; #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale&amp;nbsp; 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&amp;nbsp; 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&amp;nbsp; Co 105-420&lt;/p&gt;</td></tr></tbody></table>

physiology lab demonstration: glomerular filtration rate in a rat

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.

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...

Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

生理学实验室演示：大鼠肾小球滤过率 |生物学 |视频

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

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.

生理学实验室演示:肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

physiology-lab-demonstration-glomerular-filtration-rate-in-a-rat

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Osmoregulation and Excretion

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17.3K 次观看.  U.S. Army Institute of Surgical Research. 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. 

视频: Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

科研

医学

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

生物学

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

高灵敏度测量与氟素等西洋盐-聚糖小鼠的球状渗透性 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).