وكان مصدر التمويل للمظاهرة مختبر منحة NIGMS: GM077119. نشكر الدكتور جوزيف R. هايوود والدكتور بيتر كوبيت على دعمهم للتتسبب قصيرة في التكاملية ونظم الجهاز الصيدلة. كما نشكر السيدة هانا جارفر لها الدعم الفني للمظاهرة المختبر.

قبل الشروع في أي إجراء الحيوان، لجنة رعاية الحيوان واستخدام المؤسسية (IACUC) يجب الموافقة على البروتوكول. وقد وافق هذا البروتوكول من قبل جامعة IACUC ولاية ميشيغان. 1. إعداد قبل مختبر FITC-حبوب الحل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الدافئة 20 مل من المياه المالحة إلى 70 درجة مئوية، وببطء ضجة في 100 ملغ من FITC-حبوب (5 ملغ / مل FITC-حبوب) حتى يذوب كل حبوب. </li><li style=";text-align:right;direction:rtl"> حل بارد لRT وإضافة 800 ملغ من ألبومين المصل البقري (40 ملغ / مل BSA، مسحوق مجفف بالتجميد، الجلوبيولين أساسا الحرة، الذيفان الداخلي منخفضة، ≥98٪ نقاء بواسطة agarose هلام الكهربائي). </li><li style=";text-align:right;direction:rtl"> تصفية الحل حبوب-BSA مع ورق الترشيح (الصف 1). وضع حل تصفيتها في حقنة 20 مل مع فلتر حقنة طرف (0.2 ميكرون)، ويغطى بورق للحماية من الضوء. </li></ol> 2. التخدير وجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع فأر في غرفة تحريض مليئة 5٪ الأيزوفلورين للحث على التخدير. سجل بودالوزن ص (250-350 ز) ووضع فأر على منصة الجراحية ساخنة تهدف إلى الحفاظ على 37 درجة مئوية درجة حرارة الجسم في كافة مراحل التجربة. تأمين بلطف الفئران على منصة مع الشريط المختبر على الكفوف. الحفاظ على التخدير مع 1-2٪ الأيزوفلورين مع الصف الطبية 100٪ O 2 في معدل تدفق الهواء من 0،8-1،0 لتر / دقيقة. </li><li style=";text-align:right;direction:rtl"> ادخال قسطرة مدبب (داخل الأوعية OD طرف، 2.7F) في الشريان الفخذي لضغط الدم ورصد معدل ضربات القلب، وأخذ عينات الدم. </li><li style=";text-align:right;direction:rtl"> ادخال قسطرة (PE-50) في الوريد الفخذي للحبوب الحقن في الوريد. تأمين القسطرة إلى الأنسجة المحيطة مع 5-O مضفر الحرير خياطة جراحية 6. </li><li style=";text-align:right;direction:rtl"> إرفاق القسطرة الشريانية لمحول الضغط قياس الضغط. سجل ضغط الدم ومعدل ضربات القلب باستخدام برنامج الحصول على البيانات وعرضها على شاشة الكمبيوتر في الوقت الحقيقي. ويتجلى هذا الأسلوب في التفاصيل على شريط فيديو 6. </li><li style=";text-align:right;direction:rtl"> فضح المثانة عن طريق شق فوق العانة. قطع صغيرةثقب في طرف المثانة وإدراج قنية (PE-190) مع حرارة اندلع طرف داخل المثانة لجمع البول. تأمين قنية إلى المثانة مع خياطة محفظة سلسلة. </li></ol> 3. البول وجمع الدم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع حقنة من FITC-حبوب في ضخ حقنة مع معدل تدفق مجموعة من 1 مل / ساعة لكل 100 غرام من وزن الجسم (3 مل / ساعة لفأر وزنها 300 غرام). نعلق حقنة لقسطرة الوريد الفخذي. بدء ضخ حبوب والسماح لفترة موازنة 2/1 ساعة. الحفاظ على حقنة مغطاة بورق للحماية من الضوء. </li><li style=";text-align:right;direction:rtl"> تحديد ما إذا كان معدل تدفق البول هو ثابت وكاف لتحليل العينة (20 ميكرولتر / دقيقة) عن طريق جمع عينة البول في وزنه قبل القارورة جمع لمدة 10 دقيقة. تحديد حجم البول gravimetrically مع مقياس رقمي. وحدة تخزين البول الكافي لفترة التحصيل 10 دقيقة 0.2 مل. مواصلة جمع عينات البول حتى تشير إلى مجموعتين متتاليتين معدل تدفق البول من 20 ميكرولتر / مأو أكثر. </li><li style=";text-align:right;direction:rtl"> عينات ما قبل الدواء <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع عينة البول خلال فترة 20 دقيقة. جمع عينة الدم (0.5 مل) من القسطرة الشريانية في منتصف فترة جمع البول. كن حذرا لواضحة تماما القسطرة الشريانية من المياه المالحة قبل جمع عينة الدم في قارورة جمع تحتوي على 1 U الهيبارين. استخدام قارورة جمع مع علامات حجم لتسهيل جمع 0.5 مل من الدم الشرياني. </li><li style=";text-align:right;direction:rtl"> طرد القسطرة الشريانية مع الهيبارين المالحة (20 U / مل) لمسح القسطرة من الدم (حوالي 0.1 مل). يجب أن يكون طول القسطرة الشريانية قصيرة قدر الإمكان للحد من حجم الهيبارين المالحة المطلوبة للقضاء. ملاحظة: عينات الدم المخفض تنتج حسابات غير دقيقة من GFR وإفراز كسور من نا وK. </li><li style=";text-align:right;direction:rtl"> انتظر 10 دقيقة، وتكرار مجموعة من ثانية البول قبل المخدرات وعينة من الدم. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد جمع عينتين قبل المخدرات وإدارة ودرو مدر للبول<html> ز، فوروسيميد (10 ملغ / كلغ)، عن طريق القسطرة في الشرايين. طرد القسطرة الشريانية مع المياه المالحة heparinized لمسح القسطرة من المخدرات. الحرص على منع ضخ الهواء عن طريق القسطرة في الشرايين. تسجيل الوقت من حقن فوروسيميد. </li><li style=";text-align:right;direction:rtl"> عينات بعد المخدرات: في كل نقطة من النقاط الزمنية 3 أدناه، وجمع عينة من البول خلال فترة جمع 10 دقيقة، وعينة الدم (0.5 مل) في منتصف فترة جمع البول. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> العينة بعد المخدرات 1 - جمع خمسة دقيقة بعد فوروسيميد. </li><li style=";text-align:right;direction:rtl"> العينة بعد المخدرات 2 - جمع عشرة دقيقة بعد فوروسيميد. </li><li style=";text-align:right;direction:rtl"> العينة بعد المخدرات 3 - جمع خمسة عشر دقيقة بعد فوروسيميد. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد الانتهاء من جمع كل العينات، الموت ببطء الفئران وفقا للإجراءات المؤسسية بضع الصدر وإزالة القلب. إزالة كل من الكليتين. Decapsulate (إزالة الغشاء المحيط) وصمة عار الكلى لإزالة الدم الزائد. وزن الكلى. </li></ol>الحمار = &quot;jove_title&quot;&gt; 4. تحليل عينة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قياس كافة وحدات التخزين عينة البول gravimetrically مع مقياس رقمي، وأوزان قياسية. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي عينات الدم الكامل مع جهاز للطرد المركزي فوق المنضدة (1800 x ج) لفصل البلازما. نقل عينات البلازما إلى قارورة صغيرة المسمى. </li><li style=";text-align:right;direction:rtl"> تحليل تركيزات البوتاسيوم والصوديوم في عينات البول والبلازما مع محلل الصوديوم / البوتاسيوم. </li><li style=";text-align:right;direction:rtl"> قياس FITC-حبوب في البلازما والبول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع البول قبل المخدرات (من 1: 200-1: 400)، وبول بعد المخدرات (1:10) مع HEPES العازلة (500 ملي، ودرجة الحموضة 7.4). </li><li style=";text-align:right;direction:rtl"> إضافة 40 ميكرولتر من ميكرولتر القياسية أو عينة و 60 العازلة HEPES في 96 لوحة جيدا (عينة واحدة لكل بئر) والسماح لخلط لمدة 10 دقيقة في حين مغطاة بورق الألمنيوم. </li><li style=";text-align:right;direction:rtl"> توليد منحنى القياسية لFITC-حبوب لتركيزات 6.25، 12.5، 25، 50، 100، 200، 400 ميكروغرام / مل (الشكل 1). تحديد مضان FITC-حبوب في العينات والمعايير باستخدام قارئ صفيحة ميكروسكوبية مع السابقينالاقتباس وانبعاث موجات من 485 و 538 نانومتر، على التوالي. </li><li style=";text-align:right;direction:rtl"> تناسب القيم الفلورسنت لمعايير ل4-paramter ظيفة اللوجستية تحليل الانحدار. وتستخدم المعلمات وظيفة الانحدار لحساب تركيز FITC-حبوب في البلازما والبول عينات (الجدول 1). </li></ol></li></ol> 5. تحليل ما بعد المختبر من النتائج: الحسابات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حساب معدل تدفق البول (UV، مل / دقيقة): [حجم البول جمع (مل)] ÷ [وقت جمع (دقيقة)] </li><li style=";text-align:right;direction:rtl"> حساب معدل الترشيح الكبيبي (GFR، مل / دقيقة): [البول تركيز حبوب (ميكروغرام / مل) × الأشعة فوق البنفسجية (مل / دقيقة)] ÷ [البلازما حبوب اضرب. (ميكروغرام / مل)] </li><li style=";text-align:right;direction:rtl"> حساب المصفاة تحميل الصوديوم (ميكرومول / دقيقة): البلازما تركيز الصوديوم (ميكرومول / لتر) س GFR (مل / دقيقة) </li><li style=";text-align:right;direction:rtl"> حساب الصوديوم إفراز معدل (U V نا؛ ميكرومول / دقيقة): البول تركيز الصوديوم (ميكرومول / لتر) × الأشعة فوق البنفسجية (مل / دقيقة) </li><li style=";text-align:right;direction:rtl"> حساب كسور إفراز الصوديوم (FE نا؛٪): [U V نا (ميكرومول / دقيقة)] ÷ [تصفيتها تحميل الصوديوم (ميكرومول / دقيقة)] × 100 </li><li style=";text-align:right;direction:rtl"> حساب تحميل المصفاة البوتاسيوم (ميكرومول / دقيقة): البلازما تركيز البوتاسيوم (ميكرومول / لتر) × GFR (مل / دقيقة) </li><li style=";text-align:right;direction:rtl"> حساب البوتاسيوم إفراز معدل (U K V؛ ميكرومول / دقيقة): البول تركيز البوتاسيوم (ميكرومول / لتر) × الأشعة فوق البنفسجية (مل / دقيقة) </li><li style=";text-align:right;direction:rtl"> حساب كسور إفراز البوتاسيوم (K FE؛٪): [U K V (ميكرومول / دقيقة)] ÷ [تصفيتها البوتاسيوم تحميل (ميكرومول / دقيقة)] × 100 </li></ol>

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تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة. آراء أو التأكيدات الواردة في هذه الوثيقة هي آراء خاصة للمؤلف، وليس يمكن تفسيره بأنه مسؤول أو يعكس وجهات نظر قسم من الجيش أو وزارة الدفاع.

علامة المناسبة لقياس GFR يجب أن تفي أربعة معايير هي: أن تتم تصفيته بحرية في الكبيبة، تكون غير منضم إلى بروتينات البلازما، ولا يمكن استيعابها ولا يفرز في كليون. حبوب هو بوليمر الفركتوز الذي يرضي هذه المعايير. ونتيجة لذلك، يعتبر التصفية الكلوية للحبوب المعيار الذهبي لقي...

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.

كان مدر للبول يستخدم في المظاهرة مختبر فوروسيميد الذي يثبط بشكل سريع جدا استيعاب من البوتاسيوم والصوديوم تصفيتها عن طريق الكلى مما أدى إلى زيادة الصوديوم، البوتاسيوم، وإفراز المياه في غضون دقائق من الدواء. بواسطة آلية الأساسية، يجب أن يكون فوروسيميد الحد الأدنى من ...

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

عرض مختبر علم وظائف الأعضاء: معدل الترشيح الكبيبي في الجرذ | الأحياء | فيديو

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.

علم وظائف الأعضاء مختبر مظاهرة: معدل الترشيح الكبيبي في الجرذ

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 Views.  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. 

Video: 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.

قياس نفاذية كبيبي الجزيئات نيون باستخدام المجهر 2 فوتون في ميونيخ فئران ويستار

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

Medicine

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.

تقييم عبر الجلد من وظيفة الكلى في القوارض واعية

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.

تقييم لوظيفة الكلي في نماذج الماوس من المرض الكبيبي

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.

وهناك طريقة عالية الإنتاجية لقياس معدل الترشيح الكبيبي في الفئران وإدراكا

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

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.

طريقة سيفينج فعالة لعزل Glomeruli سليمة من الفئران الكبار الكلي

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.

قياس معدل الترشيح الكبيبي في الفئران عبر الجلد

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.

قياس حساس للغاية من نفاذية الكبيبي في الفئران مع الفلورسيين إيزوثيوسيانات-بوليسوكروز 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.

على قناة واحدة وتحليل الكالسيوم التصوير في Podocytes من الكبيبات المعزولة حديثا

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.

استخدام الفحص المجهري 2-فوتون لتحديد آثار انسداد الحالب المزمن من جانب واحد على العمليات الكبيبية

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).