Fysiologi Lab Demonstration: Glomerular Filtration Rate i en råtta
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
En lämplig markör för GFR mätning måste uppfylla fyra kriterier: fritt filtreras vid glomerulus, vara obundet till plasmaproteiner, och varken absorberas eller utsöndras i nephron. Inulin är en fruktospolymer som uppfyller dessa kriterier. Som en följd av detta njurclearance av inulin ansett guldnormen för att mäta GFR 7. Den visade tekniken representerar den traditionella tillvägagångssättet för att fastställa renal utsöndring av inulin med hjälp av tidsurinsamlingar under en konstant infusi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Den finansieringskälla för labbet demonstrationen var NIGMS bidrag: GM077119. Vi tackar Dr Joseph R. Haywood och Dr Peter Cobbett för deras stöd av Short Couse i integrativ och orgel Systems farmakologi. Vi vill också tacka Ms. Hannah Garver för henne tekniskt stöd från laboratorie demonstration.
Materials
| 5-0 Braided Silk Surgical Suture | Surgical Specialties Corp | SP1033 | |
| Assay Plate, 96-Well | Costar | 3922 | |
| Bovine Serum Albumin | Sigma Chemical Co | A2934-25G | |
| Centrifuge | Beckman Coulter | MicroFuge 18, 357160 | |
| Conical Sample Tubes | Dot Scientific Inc. | #711-FTG | |
| Cotton Tipped Applicators | Solon Manufacturing Co | 56200 | |
| Data Acquisition Software | ADInstruments | LabChart Pro 7.0 | |
| Digital Scale | Denver Instrument | APX-4001 | |
| FITC-Inulin | Sigma Chemical Co | F3272-1G | |
| Gauze Sponges | Covidien | 2146 | |
| Heated Surgical Bed | EZ-Anesthesia | EZ-212 | |
| Heparin | Sagnet | NDC 25021-402-10 | |
| HEPES | Sigma Chemical Co | H3375 | |
| Isoflurane | Abbott Animal Health | IsoFlo, 5260-04-05 | |
| Isoflurane Vaporizer | EZ-Anesthesia | EZ-190F | |
| Micro Dissecting Forceps | Biomedical Research Instruments Inc. | 70-1020 | |
| Microplate Reader – Fluoroskan | ThermoScientific | Ascent FL, 5210460 | |
| NOVA 5+ Sodium/Potassium Analyzer | NOVA BioMedical | 14156 | |
| Olsen-Hegar Needle Holders with Scissors | Fine Science Tools | 12002-12 | |
| PE-190 (for bladder catheter) | BD Medical | 427435 | |
| Pressure Transducer | ADInstruments | MLT1199 | |
| Pyrex Culture Tubes | Corning Inc. | 99445-12 | |
| Rat Femoral Tapered Artery Catheter | Strategic Applications Inc. | RFA-01 | |
| Salix Furosemide 5% | Intervet | #34-478 | |
| Strabismus Scissors | Fine Science Tools | 14075-11 | |
| Student Surgical Scissors | Fine Science Tools | 91402-12 | |
| Surgical Gloves | Kimberly-Clark | Sterling Nitrile Gloves | |
| Syringe pump | Razel Scientific | R99-E | |
| Tissue Forceps | Fine Science Tools | 91121-12 | |
| Tissue Scissors | George Tiemann Co | 105-420 |
5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc. #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader – Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann Co 105-420
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