מקור המימון להפגנת המעבדה היה מענק NIGMS: GM077119. אנו מודים לד&quot;ר יוסף ר &#39;הייווד וד&quot;ר פיטר קובט על תמיכתם של couse הקצר באינטגרטיבית ואיברי מערכות פרמקולוגיה. כמו כן, אנו מודים לגב &#39;חנה Garver לתמיכה הטכנית שלה הפגנת המעבדה.

לפני כל הליך בעלי חיים, טיפול בבעלי החיים הוועדה המוסדית ושימוש (IACUC) חייבת לאשר את הפרוטוקול. פרוטוקול זה אושר על ידי IACUC אוניברסיטת מדינת מישיגן. 1. הכנת מעבדה-טרום של פתרון FITC-אינולין <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 20 מיליליטר של תמיסת מלח חם עד 70 מעלות צלזיוס ולאט לאט מוסיפים של FITC-אינולין (5 מ&quot;ג / מיליליטר FITC-אינולין) 100 מ&quot;ג עד שכל אינולין נמס. </li><li style=";text-align:right;direction:rtl"> פתרון מגניב RT ולהוסיף 800 מ&quot;ג של אלבומין בסרום השור (40 מ&quot;ג / מיליליטר BSA, אבקת lyophilized, בעצם גלובולין רעלן פנימי ללא תשלום, נמוך, טוהר ≥98% על ידי ג&#39;ל אלקטרופורזה agarose). </li><li style=";text-align:right;direction:rtl"> סנן את פתרון אינולין-BSA עם נייר סינון (כיתה 1). מניחים את הפתרון המסונן במזרק 20 מיליליטר עם מסנן מזרק-קצה (0.2 מיקרומטר) ומכסה בנייר כסף כדי להגן מפני אור. </li></ol> 2. הרדמה וניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את החולדה בתא אינדוקציה מלא isoflurane 5% כדי לגרום להרדמה. שיא דירקטוריוןמשקל y (250-350 ז) ומניח את החולדה על פלטפורמת ניתוח מחוממת נועדה לשמור על 37 מעלות צלזיוס טמפרטורת גוף לאורך כל הניסוי. בעדינות לאבטח את החולדה לפלטפורמה עם קלטת מעבדה על כפות. לשמור על הרדמה עם isoflurane 1-2% עם הכיתה רפואית O 100% 2 בקצב זרימת אוויר של .8-1.0 L / min. </li><li style=";text-align:right;direction:rtl"> הכנס קטטר מחודד (OD תוך-הקצה, 2.7F) לתוך עורק הירך ללחץ דם וניטור קצב לב, ודגימת דם. </li><li style=";text-align:right;direction:rtl"> הכנס קטטר (PE-50) לוריד הירך לעירוי אינולין. אבטח את הצנתר לרקמות עם תפר כירורגי משי קלוע 5-O 6. </li><li style=";text-align:right;direction:rtl"> צרף את צנתר העורקים למתמר לחץ מד לחץ. לחץ שיא דם וקצב לב באמצעות תוכנת רכישת נתונים ותצוגה על מסך מחשב בזמן אמת. טכניקה זו באה לידי ביטוי בפירוט על וידאו 6. </li><li style=";text-align:right;direction:rtl"> לחשוף את שלפוחית ​​השתן דרך חתך suprapubic. חותך קטןחור בקצה של שלפוחית ​​השתן ולהכניס צינורית (PE-190) עם חום התלקח קצה בתוך שלפוחית ​​השתן לאיסוף שתן. אבטח את הצינורית לשלפוחית ​​השתן עם תפרים טבעתיים. </li></ol> 3. תנו ודם אוסף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את המזרק של FITC-אינולין במשאבת מזרק עם שיעור זרימת סט של 1 מיליליטר / שעה למשקל גוף 100 גרם (3 מיליליטר / שעה לעכברוש במשקל 300 גרם). צרף את המזרק לקטטר וריד הירך. התחל עירוי אינולין ולאפשר תקופת איזון 1-2 שעות. שמור מזרק מכוסה בנייר כסף כדי להגן מפני אור. </li><li style=";text-align:right;direction:rtl"> לקבוע אם זרימת שתן השיעור יציב ומתאים לניתוח מדגם (20 μl / min) על ידי איסוף דגימת שתן בבקבוקון אוסף-שקל מראש לתקופה של 10 דקות. לקבוע נפח שתן gravimetrically עם סולם דיגיטלי. נפח שתן נאות לתקופה אוסף 10 דקות הוא 0.2 מיליליטר. תמשיך לאסוף דגימות שתן עד שני אוספים רצופים מצביעים זרימת שתן שיעור של 20 μl / מ &#39;באו יותר. </li><li style=";text-align:right;direction:rtl"> דוגמאות טרום תרופות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף דגימת שתן במהלך תקופה של 20 דקות. לאסוף דגימת דם (0.5 מיליליטר) מצנתר העורקים בנקודת האמצע של תקופת האיסוף שתן. היזהר ברור לחלוטין צנתר העורקים של תמיסת מלח לפני איסוף דגימת דם בבקבוקון המכיל אוסף 1 הפרין U. השתמש בקבוקוני אוסף עם סימוני נפח כדי להקל על האוסף של 0.5 מיליליטר של דם עורקים. </li><li style=";text-align:right;direction:rtl"> רוקן את הקטטר עורקים עם הפרין-מלוח (20 U / ml) כדי לנקות את הקטטר של דם (כ. 0.1 מיליליטר). אורכו של צנתר העורקים צריך להיות קצר ככל האפשר כדי להגביל את הנפח של הפרין-מלוח הדרוש כדי לשטוף. הערה: דגימות דם מדולל לייצר חישובים מדויקים של GFR והפרשה חלקית של Na וק </li><li style=";text-align:right;direction:rtl"> חכה 10 דקות, וחזור על האוסף של שתן טרום תרופה שנייה ודגימת דם. </li></ol></li><li style=";text-align:right;direction:rtl"> בעקבות האוסף של שתי דגימות טרום תרופה, לנהל DRU משתן<html> g, פורוסמיד (10 מ&quot;ג / קילוגרם), דרך צנתר העורקים. רוקן את הקטטר עורקים עם מלוחים heparinized כדי לנקות את הקטטר של תרופה. לטפל כדי למנוע את הזריקה של אוויר דרך צנתר העורקים. רשום את הזמן של הזרקת פורוסמיד. </li><li style=";text-align:right;direction:rtl"> דוגמאות פוסט-סמים: בכל אחת מנקודות הזמן 3 להלן, לאסוף דגימת שתן בתקופה אוסף 10 דקות, ודגימת דם (0.5 מיליליטר) בנקודת האמצע של תקופת האיסוף שתן. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדוגמא הודעה בסמים 1 - לאסוף חמש דקות לאחר פורוסמיד. </li><li style=";text-align:right;direction:rtl"> על הודעה בסמים לדוגמא 2 - לאסוף עשר דקות לאחר פורוסמיד. </li><li style=";text-align:right;direction:rtl"> על הודעה בסמים לדוגמא 3 - לאסוף חמישה עשר דקות לאחר פורוסמיד. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר כל הדגימות נאספו, להרדים את העכברוש בהתאם לנהלים מוסדיים על ידי פתיחת בית החזה וההסרה של הלב. הסר את שני הכליות. Decapsulate (להסיר את הקרום המקיף) ולמחוק את הכליות כדי להסיר דם עודף. לשקול את הכליות. </li></ol>התחת = &quot;jove_title&quot;&gt; 4. ניתוח מדגם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> למדוד את כל כרכי דגימת שתן gravimetrically עם סולם דיגיטלי, ומשקלי שיא. </li><li style=";text-align:right;direction:rtl"> דגימות דם כל צנטריפוגה עם צנטריפוגה בטבלה העליונה (1,800 XG) להפריד פלזמה. העבר את דגימות פלזמה לבקבוקונים קטנים שכותרתו. </li><li style=";text-align:right;direction:rtl"> לנתח ריכוזי Na ו- K בדגימות שתן ופלזמה עם מנתח נתרן / אשלגן. </li><li style=";text-align:right;direction:rtl"> מדידה של FITC-אינולין בפלזמה ובשתן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל את שתן מראש סמים (מ1: 200 עד 1: 400), ושתנו לאחר סמים (01:10) עם חיץ HEPES (500 מ&quot;מ, pH 7.4). </li><li style=";text-align:right;direction:rtl"> הוסף 40 μl של μl הסטנדרטי או מדגם ו -60 של חיץ HEPES בצלחת גם 96 (מדגם אחד לכל היטב) ולאפשר לערבב למשך 10 דקות בזמן מכוסה ברדיד אלומיניום. </li><li style=";text-align:right;direction:rtl"> ליצור עקומה סטנדרטית עבור FITC-אינולין לריכוזים של 6.25, 12.5, 25, 50, 100, 200, 400 מיקרוגרם / מיליליטר (איור 1). לקבוע הקרינה FITC-אינולין בדגימות וסטנדרטים באמצעות קורא microplate עם האקסאורכי גל ציטוט ופליטה של ​​485 ו538 ננומטר, בהתאמה. </li><li style=";text-align:right;direction:rtl"> התאם את ערכי ניאון לסטנדרטים לניתוח רגרסיה פונקציה לוגיסטית 4 paramter. הפרמטרים פונקצית רגרסיה משמשים לחישוב ריכוז FITC-אינולין בדגימות פלזמה ובשתן (טבלה 1). </li></ol></li></ol> ניתוח מעבדה ההודעה 5. תוצאות: חישובים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחשב את שיעור זרימת שתן (UV; מיליליטר / דקה): [נפח השתן שנאסף (מיליליטר)] ÷ [זמן של אוסף (דקות)] </li><li style=";text-align:right;direction:rtl"> לחשב גלומרולרי סינון שיעור (GFR; מיליליטר / דקה): [ריכוז שתן אינולין (מיקרוגרם / מיליליטר) x UV (מיליליטר / דקה)] ÷ [קונצרט כלשהו אינולין פלזמה. (מיקרוגרם / מיליליטר)] </li><li style=";text-align:right;direction:rtl"> לחשב טען נתרן מסונן (μmol / min): ריכוז נתרן פלזמה GFR (μmol / מיליליטר) x (מיליליטר / דקה) </li><li style=";text-align:right;direction:rtl"> לחשב נתרן הפרשה שיעור (U Na V; μmol / min): ריכוז נתרן שתן (μmol / מיליליטר) x UV (מיליליטר / דקה) </li><li style=";text-align:right;direction:rtl"> לחשב שברי הפרשה של נתרן (Na FE;%): [U Na V (μmol / min)] ÷ [טען נתרן מסונן (μmol / min)] x 100 </li><li style=";text-align:right;direction:rtl"> לחשב טען מסונן אשלגן (μmol / min): ריכוז אשלגן פלזמה (μmol / מיליליטר) x GFR (מיליליטר / דקה) </li><li style=";text-align:right;direction:rtl"> לחשב אשלגן הפרשה שיעור (U K V; μmol / min): ריכוז אשלגן שתן (μmol / מיליליטר) x UV (מיליליטר / דקה) </li><li style=";text-align:right;direction:rtl"> לחשב שברי הפרשה של אשלגן (FE K;%): [U K V (μmol / min)] ÷ [טען אשלגן מסונן (μmol / min)] x 100 </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+filtration+rate+determinations+in+conscious+type+II+diabetic+mice.">Glomerular filtration rate determinations in conscious type II diabetic mice.</a> Americam Journal of Physiology. 300 (3), F618-F625 (2011).</li><li>Rosen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anaesthesia+and+surgery+on+renal+hemodynamics.">Effects of anaesthesia and surgery on renal hemodynamics.</a> British Journal of Anesthesiology. 44, 252-258 (1972).</li><li>Cousins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+the+kidney.">Anesthesia and the kidney.</a> Anaesthesia and intensive care. 11 (4), 292-320 (1983).</li><li>Walter, S. J., Zewde, T., Shirley, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+anaesthesia+and+standard+clearance+procedures+on+renal+function+in+the+rat.">The effect of anaesthesia and standard clearance procedures on renal function in the rat.</a> Quarterly Journal of Experimental Physiology. 74, 805-812 (1989).</li><li>Rieg, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> Journal of Visualized Experiments. (75), (2013).</li></ol>

המחברים מצהירים שאין להם אינטרסים כלכליים מתחרים. הדעות או הטענות הכלולות במסמך זה הן את הדעות פרטיות של המחבר ואינם להתפרש רשמי או כמשקפים את הדעות של המחלקה לצבא או משרד ביטחון.

סמן מתאים למדידת GFR לעמוד בארבעה קריטריונים: להיות מסונן באופן חופשי בglomerulus, להיות מאוגד לחלבוני פלזמה, ולא ייקלט ולא מופרש בנפרון. אינולין הוא פולימר פרוקטוז אשר עונה על קריטריונים אלה. כתוצאה מכך, הפינוי כלייתי של אינולין נחשב תקן הזהב למדידת GFR 7. הטכניקה הפגי?...

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.

משתן משמש בהפגנת המעבדה היה פורוסמיד אשר מעכב את הספיגה של של Na ו- K המסוננת על ידי הכליות וכתוצאה מכך גדל Na, K, והפרשת מים בתוך דקות של מתן תרופה מהר מאוד. על ידי המנגנון העיקרי שלה, פורוסמיד צריך להיות השפעות מינימליות על GFR והעומס המסונן של Na ו- K, אבל יגביר את זרימת שתן, ?...

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

הדגמת מעבדת פיזיולוגיה: קצב סינון גלומרולרי בחולדה | ביולוגיה | וִידֵאוֹ

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.

הפגנת מעבדה לפיזיולוגיה: גלומרולרי סינון שיעור בחולדה

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.

כימות חדירות גלומרולרי של מקרומולקולות פלורסנט באמצעות מיקרוסקופ 2-פוטון בחולדות Wistar מינכן

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.

הערכת Transcutaneous של תפקוד כלייתי במכרסמי Conscious

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.

הערכה של תפקוד כליות במודלים של העכבר של מחלת Glomerular

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.

שיטת תפוקה גבוהה למדידה של קצב סינון הגלומרולרי בעכברים בהכרה

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.

דרך יעילה Sieving כדי לבודד Glomeruli ללא פגע מן החולדה למבוגרים כליות

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.

Transdermal מדידה של קצב הסינון Glomerular בעכברים

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.

מדידה רגישה מאוד של חדירות הפקאוליות בעכברים עם פלואורואסעין איזוטיקריאט-פוליסוכרוז 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.

ערוץ יחיד הדמיה ניתוח וסידן בPodocytes של glomeruli מבודדת טרי

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.

שימוש במיקרוסקופיה של 2-פוטונים כדי לכמת את ההשפעות של חסימת שופכה חד-צדדית כרונית על תהליכים גלומרולריים

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).