GM077119：ラボのデモンストレーションのための資金源は、NIGMS助成金でした。我々は、統合および臓器システム薬理学におけるショートクーゼの彼らのサポートのために博士ジョセフ·R·ヘイウッドとピーター·コベットに感謝します。また、実験室でのデモの彼女の技術支援のためにさんハンナGarverに感謝します。

任意の動物手順、制度的動物のケアと使用委員会（IACUC）の前には、プロトコルを承認する必要があります。このプロトコルは、ミシガン州立大学IACUCによって承認されました。 FITC-イヌリン溶液の1前のラボの準備<ol><li>全てのイヌリンが溶解するまでゆっくりと加温20〜70℃の生理食塩水のミリリットルとは、FITC-イヌリン（5 mg / mlのFITC-イヌリン）を100mgを加えて混ぜます。 </li><li>室温に冷却液と（基本的に、アガロースゲル電気泳動によりフリー、低エンドトキシン、≥98％純度グロブリン、40 mg / mlのBSA、凍結乾燥粉末）、ウシ血清アルブミン800mgのを追加します。 </li><li>濾紙（グレード1）とイヌリンBSA溶液をフィルタリングします。シリンジチップフィルター（0.2μm）で、20 mlシリンジで濾過した溶液を置き、光から保護するためにホイルでカバーしています。 </li></ol> 2.麻酔と手術<ol><li>麻酔を誘導するために、5％のイソフルランで満たされた導入室にラットを置きます。レコードBODY重量（250〜350グラム）と、実験を通して37℃の体温を維持するように設計された加熱された外科用プラットフォーム上にラットを置きます。ゆっくり足の上に実験用テープでプラットフォームにラットを固定します。 0.8〜1.0リットル/分の空気流量で医療グレード100％のO 2を1〜2％イソフルランで麻酔を維持します。 </li><li>血圧および心拍数のモニタリング、及び血液サンプリングのために大腿動脈にカテーテルテーパー（血管内先端OD、2.7F）を挿入します。 </li><li>イヌリン注入のための大腿静脈にカテーテル（PE-50）を挿入します。 5-O編み絹縫合糸6と周囲の組織にカテーテルを固定します。 </li><li>ひずみゲージ圧力変換器に動脈カテーテルを取り付けます。リアルタイムでコンピュータ画面上のデータ取得ソフトウェア、ディスプレイを使用して記録する血圧と心拍数。この技術は、映像6に詳細に示されています。 </li><li>恥骨上切開により膀胱を公開します。小さなをカット膀胱の先端に穴や熱でカニューレ（PE-190）を挿入するには、尿採取のために、膀胱の内側に先端をフレア。巾着縫合で膀胱にカニューレを固定します。 </li></ol> 3.尿や血液コレクション<ol><li> 1ミリリットル/体重（300グラムの重さのラットについて3ミリリットル/時）の100グラムあたりの時間の流速が設定されたシリンジポンプにFITC-イヌリンの注射器を置きます。大腿静脈カテーテルに注射器を取り付けます。イヌリンの注入を開始し、1〜2時間の平衡期間を可能にします。光から保護するために箔で覆われた注射器を保管してください。 </li><li>尿流量が安定しており、10分の期間、予め秤量した回収バイアル中の尿サンプルを採取することにより、試料分析（20μL/分）のために適切であるかどうかを確認します。デジタルスケールで重量測定により尿量を決定します。 10分の回収期間のための適切な尿量は、0.2 mlです。二つの連続コレクションが20μL/ Mの尿流量を示すまでの尿サンプルを収集し続けますで以上。 </li><li>プレドラッグサンプル<ol><li> 20分の期間中に尿サンプルを採取します。尿収集期間の中間点において動脈カテーテルから血液サンプル（0.5ml）に収集します。 1 Uのヘパリンを含むコレクションバイアルに血液サンプルを収集する前に生理食塩水を完全に透明に動脈カテーテルに注意してください。動脈血の0.5ミリリットルの収集を容易にするために、ボリュームマーキングコレクションバイアルを使用します。 </li><li>血液（約0.1ミリリットル）のカテーテルをクリアするためにヘパリン生理食塩水（20 U / ml）で動脈カテーテルをフラッシュします。動脈カテーテルの長さは、洗い流すために必要なヘパリン - 生理食塩水の量​​を制限することが可能な限り短くすべきです。 注：希釈された血液サンプルはGFRおよびNaとKの分数排泄の不正確な計算を生成します</li><li> 10分待ってから、第2のプリ薬物の尿と血液サンプルの収集を繰り返します。 </li></ol></li><li> 2つの事前薬物サンプルの収集に続いて、利尿DRUの管理G、フロセミド（10mg / kgの）、動脈カテーテルを介し。薬物のカテーテルをクリアするためにヘパリン化生理食塩水で動脈カテーテルをフラッシュします。動脈カテーテルを介して空気を注入しないように注意してください。フロセミド注射の時間を記録します。 </li><li>尿収集期間の中間点で下3時間点の各々で、10分間の収集期間中に尿サンプルを採取し、血液試料（0.5mL）を：ポスト薬剤サンプル。 <ol><li>後薬物サンプル1について - フロセミド後5分を収集します。 </li><li>後薬物のサンプル2について - フロセミド後10分を集めます。 </li><li>後薬物サンプル3について - フロセミド後に15分を集めます。 </li></ol></li><li>全てのサンプルが収集された後、開胸し、心臓を除去することによって機関の手順に従ってラット安楽死させます。両方の腎臓を削除します。逆カプセル化（周囲の膜を除去）し、過剰の血液を除去するために腎臓をブロット。腎臓を計量。 </li></ol><ol><li>デジタルスケールで重量測定により、すべての尿サンプル量を測定し、記録の重み。 </li><li>テーブルトップ遠心機（1,800 XG）で遠心分離し、全血サンプルは、血漿を分離しました。小さなラベル付きバイアルに血漿サンプルを転送します。 </li><li>ナトリウム/カリウムアナライザーで尿および血漿試料中のNa、Kの濃度を分析します。 </li><li>血漿および尿中のFITC-イヌリンの測定<ol><li> HEPES緩衝液（500mMの、pH7.4）で、ポスト薬物尿（1:10）（400：1：200〜1）から予め薬物尿を希釈します。 </li><li> 96ウェルプレート（ウェルごとに1つのサンプル）にHEPES緩衝液の標準またはサンプルを60μlの40μlを添加して、アルミホイルで覆いながら10分間混合することができます。 </li><li>の濃度についてのFITC-イヌリンの標準曲線を生成6.25、12.5、25、50、100、200、400 / mlの（ 図1）。元にマイクロプレートリーダーを使用してサンプルと標準でFITC-イヌリン蛍光を決定それぞれ485および538 nmでの引用及び発光波長。 </li><li> 4-PARAMTERロジスティック関数の回帰分析に標準の蛍光値を取り付けます。回帰関数のパラメータは、血漿および尿サンプル（ 表1）にFITC-イヌリン濃度を計算するために使用されます。 </li></ol></li></ol>結果5.ポスト​​ラボ分析：計算<ol><li> 【尿量収集（ミリリットル）]÷[収集の時間（分）]：;尿流量（ml /分UV）を計算します</li><li>糸球体濾過率を計算（GFR、ml /分）：尿イヌリン濃度（/ mlの）UV（ml /分）をX]÷[血漿イヌリン濃濃度（μg/ ​​ml）を] </li><li>計算フィルタリングさナトリウム負荷（μモル/分）：血漿ナトリウム濃度（モル/ ml）をGFR（ml /分）をxは</li><li>尿中ナトリウム濃度（モル/ ml）をUV（ml /分）をX：;ナトリウム排泄率（μモル/分U のNa V）を計算します</li><li>ナトリウム排泄分画（;％FE NA）を計算します：[U のNa V（マイクロモル/分）]÷[濾過ナトリウム負荷（マイクロモル/分）]×100 </li><li>計算フィルタリングさカリウム負荷（μモル/分）：血漿カリウム濃度（モル/ ml）をGFR（ml /分）をxは</li><li>尿中カリウム濃度（モル/ ml）をUV（ml /分）をX：;カリウム排泄率（μモル/分U K V）を計算します</li><li>計算カリウムの分別排泄（FE K;％）：[U K V（マイクロモル/分）]÷[濾過カリウムロード（マイクロモル/分）]×100 </li></ol>

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</ol>

著者は、彼らが競合する金融利害関係がないことを宣言します。ここに含まれる意見やアサーションは、作者の個人ビューであり、公式として、または陸軍省や国防総省の見解を反映するものとして解釈されるべきではありません。

血漿タンパク質に結合していないこと、自由に糸球体で濾過され、吸収されたことも、ネフロンに分泌されることもない：GFR測定のための適切なマーカーは、4つの基準を満たす必要があります。イヌリンはこれらの基準を満たすフルクトースポリマーです。その結果、イヌリンの腎クリアランスはGFR 7を測定するためのゴールドスタンダードと考えられています。実証技術はイヌリン8,9の一定の注入時に時限尿コレクションを使用して、イヌリンの腎クリアランスを決定する従来のアプローチを表しています。従来のイヌリンの測定は、分光光度計10,11によって測定されたイヌリンの定量比色決意を生成するためにアントロン法を用いて作製されています。しかし、尿および血漿の小さい体積でイヌリンの測定を容易にするための試みにおいて、イヌリンは15-17 12-14放射性、蛍光標識でタグ付けされています。このビデオUに提示ラボデモSED FITCが原因人間の放射線被曝の危険性の欠如とFITC蛍光15の測定の容易さの腎機能を測定するためのイヌリンを標識しました。 このラボでのデモは、最小限の実験室でのスキルを持つ学生に腎機能を測定する方法の概念理解を提供することを意図しています。したがって、FITC-イヌリン溶液、及び動物の外科的準備の前ラボ製剤は、デモンストレーションの開始前に経験のある技術者によって行われます。学生は1〜2時間のイヌリンの平衡期間の終了時にデモに到着します。この時点で、学生は事前ラボ概要が提示され、動物に行われている手順を知らさ。二人の学生は、1動物実験に割り当てられ、前と利尿薬の投与後、血液および尿サンプルを採取する方法で指示されています。血液および尿サンプルの分析はエクスペリエンス3によって行われますNCED技術者との結果が腎機能の計算のための学生に配信されます。結果は、デモンストレーションの後にスケジュールすることができるポスト·ラボの議論の際に提示されています。 有効回答を保証するプロトコル内のいくつかの重要なステップがあります。まず、FITCイヌリンが完全に溶解し、動物の投与前にフィルタリングされなければなりません。理想的には、FITCのイヌリンは、残留未結合のFITCを除去するために、室温で48時間水中で透析されるべきです。第二に、血漿試料は、生理食塩水の自由でなければなりません。学生は、動脈カテーテル内の生理食塩水のすべてが追放された後にのみ、血液サンプルを採取するように指示されており、唯一の血液がカテーテルの外に流れています。生理食塩水で希釈された血液試料は、血漿イヌリン、ナトリウムおよびカリウムのために不正確な値を提供します。第三に、尿の流れは、分析のために十分なサンプルを生成するために安定した適切なものでなければなりません。それの指標であるので、ベースラインでの定常尿流量が重要です安定した実験準備。尿流が低すぎると、イヌリンの注入速度は、前のサンプルのコレクションにすることができます。しかし、イヌリンの注入は、実験の過程で一定でなければならない、 すなわち 、イヌリン注入速度は、実験中に調整されるべきではありません。最後に、マイクロプレートリーダーによる血漿および尿サンプル中のイヌリン蛍光の測定は、成功した実験に重要です。マイクロプレートリーダーの仕様は、試料が希釈を必要とする場合、それはイヌリンアッセイのテストの実行がマイクロプレートリーダーの仕様を最適化し、サンプルの蛍光値であることを確実にするための努力の前にラボのデモに実施することが推奨されるかを決定しますので、標準曲線のミッドレンジ内。 考えられているゴールドスタンダードをイヌリンの腎クリアランスに基づいて、腎機能を評価するが、この技術は、動物を麻酔する必要があるため、制限があり、かつ血管と膀胱カテーテルをインストルメント。 Anesthethetic剤は、腎血行動態およびGFR 18,19に影響を与えることが示されています。しかし、イソフルランとinactinは、典型的には、腎臓19,20に対するその最小の影響による腎機能の実験に使用されています。イヌリンクリアランス技術もマウスなどの小動物に禁止することができイヌリン、複数の血液および血漿サンプルの一定の注入を必要とします。この手法の変形例は、意識のある動物におけるイヌリン21の単回注射の血漿クリアランスの測定を可能にするために開発されてきました。これらの修飾はまた、分析のための血液サンプルの小さい容積を必要とし、マウスにおいて腎機能を評価するための代替方法を提供します。 腎機能の測定は、生理学、病理学、毒物学、薬理学および疾患状態の研究にも適用可能です。腎機能のデモに参加する学生は、Tを学習しますイヌリンの腎クリアランスの彼ゴールドスタンダード技術は、腎機能を評価しました。この技術をマスターすることで、学生は腎機能の原理を理解し、それらを自分の研究に技術を適用し、手法の変更は、それらの研究のために適切であるかどうかを判断することができます。

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.

ラボのデモに使用される利尿剤は非常に迅速に薬物投与の数分以内に増加したのNa、K、その結果、腎臓、および水の排泄によってフィルタリングNaおよびKの再吸収を阻害するフロセミドました。その主要なメカニズムによって、フロセミドは、GFRおよびNa、Kのフィルタ処理負荷の最小の効果を持っている必要がありますが、尿の流れを増加し、NaおよびKの分数排泄麻酔したラットにおいて、GFRのための薬物前値の平均は3.2ミリリットル/分であったことを表3に示す代表結果、ナトリウム排泄/分0.58マイクロモル（ろ過負荷の0.1％）であり、Kの排泄は4.4でしたマイクロモル/分（ろ過負荷の27％）。フロセミド（薬物後1）、GFRおよびNa、Kのフィルタ処理負荷5分後には、影響を受けませんでした。しかし、ナトリウム排泄分画は11.5％に増加し、K排泄分画は、それぞれのフィルタ処理の負荷の63％に増加しました。 MAの測定PとHRは、フロセミドは、MAPとHR（ 表2）に最小限の影響を及ぼしたことを示しています。 実験室でのデモで評価腎機能の指標は、プラズマが、腎臓によってフィルタリングされる割合として定義GFR、ありました。ろ過NaおよびK、NaおよびKは腎臓によってフィルタリングされる割合として定義され; NaおよびKは腎臓から排泄される割合として定義NaおよびK排泄率;および腎臓から排泄されるろ過NaおよびKの割合として定義NaおよびK排泄分画、 <img alt="図1" src="/files/ftp_upload/52425/52425fig1.jpg" /> 図1：イヌリン標準曲線 FITC蛍光値は6.25、12.5、25、50、100、200および400 / mlのイヌリンを含有する標準のために示されています。 4-PARAMTERロジスティック関数の回帰分析では、ベストフィットカーブを生成します。この曲線からの回帰関数のパラメータは、FIを算出するために使用されました血漿および尿サンプル中のTC-イヌリン濃度。 <table border="0" cellpadding="0" cellspacing="0" height="515" width="592"><tbody><tr><td></td><td colspan="3"> FITC-イヌリン蛍光</td><td nowrap="nowrap">濃度</td><td></td><td>結果</td></tr><tr><td>スタンダード</td><td> 1を複製します</td><td> 2を複製</td><td>平均</td><td> / mlの</td><td>希釈</td><td> / mlの</td></tr><tr><td>ブランク</td><td align="right"> 63.9 </td><td align="right"> 64.8 </td><td align="right"> 64.4 </td><td align="right"> 0.4 </td><td align="right"> 1 </td><td align="right"> 0.4 </td></tr><tr><td> 6.25 </td><td align="right"> 253.2 </td><td align="right"> 264.1 </td><td align="right"> 258.7 </td><td align="right"> 5.9 </td><td align="right"> 1 </td><td align="right"> 5.9 </td></tr><tr><td> 12.5 </td><td align="right"> 474.0 &lt;/ TD&gt; <td align="right"> 491.3 </td><td align="right"> 482.7 </td><td align="right"> 12.5 </td><td align="right"> 1 </td><td align="right"> 12.5 </td></tr><tr><td> 25 </td><td align="right"> 854.8 </td><td align="right"> 881.3 </td><td align="right"> 868.1 </td><td align="right"> 24.4 </td><td align="right"> 1 </td><td align="right"> 24.4 </td></tr><tr><td> 50 </td><td align="right"> 1617.1 </td><td align="right"> 1618.0 </td><td align="right"> 1617.6 </td><td align="right"> 50.3 </td><td align="right"> 1 </td><td align="right"> 50.3 </td></tr><tr><td> 100 </td><td align="right"> 2813.1 </td><td align="right"> 2846.1 </td><td align="right"> 2829.6 </td><td align="right"> 101.3 </td><td align="right"> 1 </td><td align="right"> 101.3 </td></tr><tr><td> 200 </td><td align="right"> 4367.3 </td><td align="right"> 4588.7 </td><td align="right"> 4478.0 </td><td align="right"> 198.2 </td><td align="right"> 1<html></td><td align="right"> 198.2 </td></tr><tr><td> 400 </td><td align="right"> 6258.0 </td><td align="right"> 6650.0 </td><td align="right"> 6454.0 </td><td align="right"> 401.6 </td><td align="right"> 1 </td><td align="right"> 401.6 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>尿サンプル</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>プレドラッグ1 </td><td align="right"> 2443.9 </td><td align="right"> 2062.3 </td><td align="right"> 2253.1 </td><td align="right"> 88.5 </td><td align="right"> 200 </td><td align="right"> 17700 </td></tr><tr><td>プレドラッグ2 </td><td align="right"> 2266.5 </td><td align="right"> 1707.0 </td><td align="right"> 1986.8 </td><td align="right"> 76.3 </td><td align="right"> 200 </td><td align="right"> 15250 </td></tr><tr><td>ポスト薬1 </td>HT &quot;&gt; 1208.9 </td><td align="right"> 1391.2 </td><td align="right"> 1300.1 </td><td align="right"> 44.7 </td><td align="right"> 10 </td><td align="right"> 447 </td></tr><tr><td>ポスト薬2 </td><td align="right"> 2753.4 </td><td align="right"> 2120.5 </td><td align="right"> 2437.0 </td><td align="right"> 97.0 </td><td align="right"> 10 </td><td align="right"> 970 </td></tr><tr><td>ポスト薬3 </td><td align="right"> 2888.3 </td><td align="right"> 3178.0 </td><td align="right"> 3033.2 </td><td align="right"> 124.4 </td><td align="right"> 10 </td><td align="right"> 1244 </td></tr></tbody></table> 表1：イヌリンアッセイ FITC-イヌリンの蛍光値のサンプル結果は試薬ブランク、7標準、5尿サンプルのために示されています。標準およびサンプルを、二連でアッセイし、必要に応じて希釈しました。各試料の平均蛍光は、イヌリンの濃度を計算しました。<html>これらの尿試料中のイヌリン濃度は、測定値の表 （ 表2）に含まれています。 <img alt="表2" src="/files/ftp_upload/52425/52425table2.jpg" /> 表2：腎機能ラボデモンストレーション中に記録された測定値 、腎機能ラボのデモンストレーションの5の期間（2プリ薬物と3ポスト薬）中に記録された変数が正しいと左腎臓重量、平均動脈圧（MAP）、心拍数（HR）、サンプル時間、尿量、血漿及び尿中ナトリウム（Na）、カリウム（K）、及びイヌリン濃度。尿イヌリン濃度を表1に示すイヌリンアッセイから決定しました。 25table3.jpg &quot;/&gt; 表3：プロトコルセクション5に示す式を使用して記録さ測定値から計算腎機能のパラメータ 、記録された変数（ 表2）尿流量、糸球体濾過率（GFR）、GFR / gの腎臓重量、排泄速度を計算するために使用されます2つの事前薬三ポスト薬期間、ろ過負荷、及びナトリウムの分別排出し（Na）、カリウム（K）。

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

生理学実験室のデモンストレーション: ラットの糸球体濾過率 |生物学 |ビデオ

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.

生理学ラボデモンストレーション：ラットにおける糸球体濾過率

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

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17.3K ビュー.  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. 

ビデオ: 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.

ミュンヘンWistar系ラットにおける2光子顕微鏡を用いた蛍光高分子の糸球体透過性を定量化

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

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.

コンシャスげっ歯類における腎機能の経皮評価

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.

糸球体疾患のマウス ・ モデルにおける腎機能の評価

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.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

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.

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

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.

マウスにおける糸球体濾過率の経皮吸収測定

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.

フルオレセインイソチオシアネート-ポリスクロースを用いたマウスにおける糸球透過性の高感度測定 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.

新たに単離した糸球体の足細胞における単一チャネル分析とカルシウムイメージング

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.

2光子顕微鏡を使用して、糸球体プロセスに対する慢性片側尿管閉塞の影響を定量化

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).

生理学ラボデモンストレーション：ラットにおける糸球体濾過率

要約

さらに動画を探す

ミュンヘンWistar系ラットにおける2光子顕微鏡を用いた蛍光高分子の糸球体透過性を定量化

コンシャスげっ歯類における腎機能の経皮評価

糸球体疾患のマウス・モデルにおける腎機能の評価

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

マウスにおける糸球体濾過率の経皮吸収測定

フルオレセインイソチオシアネート-ポリスクロースを用いたマウスにおける糸球透過性の高感度測定 70

新たに単離した糸球体の足細胞における単一チャネル分析とカルシウムイメージング

2光子顕微鏡を使用して、糸球体プロセスに対する慢性片側尿管閉塞の影響を定量化

意識のあるラットにおける腎血流の長期連続測定

生理学ラボデモンストレーション：ラットにおける糸球体濾過率

要約

さらに動画を探す

ミュンヘンWistar系ラットにおける2光子顕微鏡を用いた蛍光高分子の糸球体透過性を定量化

コンシャスげっ歯類における腎機能の経皮評価

糸球体疾患のマウス ・ モデルにおける腎機能の評価

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

マウスにおける糸球体濾過率の経皮吸収測定

フルオレセインイソチオシアネート-ポリスクロースを用いたマウスにおける糸球透過性の高感度測定 70

新たに単離した糸球体の足細胞における単一チャネル分析とカルシウムイメージング

2光子顕微鏡を使用して、糸球体プロセスに対する慢性片側尿管閉塞の影響を定量化

意識のあるラットにおける腎血流の長期連続測定

糸球体疾患のマウス・モデルにおける腎機能の評価