Name: physiology lab demonstration: glomerular filtration rate in a rat
Uploaded: 2015-07-26T14:00:00
Description: Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

The funding source for the lab demonstration was NIGMS grant: GM077119. We thank Dr. Joseph R. Haywood and Dr. Peter Cobbett for their support of the Short Couse in Integrative and Organ Systems Pharmacology. We also thank Ms. Hannah Garver for her technical support of the laboratory demonstration.&#160;

Prior to any animal procedure, the institutional animal care and use committee (IACUC) must approve the protocol. This protocol was approved by the Michigan State University IACUC.
1. Pre-lab Preparation of FITC-inulin Solution
<ol>
	<li>Warm 20 ml of saline to 70 &#176;C and slowly stir in 100 mg of FITC-inulin (5 mg/ml FITC-inulin) until all inulin is dissolved.</li>
	<li>Cool solution to RT&#160;and add 800 mg of bovine serum albumin (40 mg/ml BSA, lyophilized powder, essentially ...

<ol>
	<li>Silverthorn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Physiology: An integrated approach. , (2012).</li><li>Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guyton and Hall Textbook of Medical Physiology. , 303-344 (2011).</li><li>Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+renal+function+in+chronic+renal+disease.">Measurement of renal function in chronic renal disease.</a> Kidney International. 38 (1), 167-184 (1990).</li><li>Thurau, K., Valtin, H., Schnermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney.">Kidney.</a> Annual Review of Physiology. 30, 441-524 (1968).</li><li>Shannon, J. A., Smith, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+excretion+of+inulin,+xylose,+and+urea+by+normal+and+phoriziniaed+man.">The excretion of inulin, xylose, and urea by normal and phoriziniaed man.</a> Journal of Clinical Investigation. 14, 393-401 (1935).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> Journal of Visualized Experiments. (59), (2012).</li><li>Sterner, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+'true'+glomerular+filtration+rate+in+healthy+adults+using+infusion+of+inulin+and+comparing+it+with+values+obtained+using+other+clearance+techniques+or+predictive+equations.">Determining 'true' glomerular filtration rate in healthy adults using infusion of inulin and comparing it with values obtained using other clearance techniques or predictive equations.</a> Scandinavian Journal of Urology and Nephrology. 42, 278-285 (2008).</li><li>Toto, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+measurement+of+renal+function+utilizing+serum+creatinine,+creatinine+clearance,+inulin+and+para-aminohippuric+acid+clearance.">Conventional measurement of renal function utilizing serum creatinine, creatinine clearance, inulin and para-aminohippuric acid clearance.</a> Current Opinion in Nephrology and Hypertension. 4 (6), 505-509 (1995).</li><li>Matavelli, L. C., Kadowitz, P. J., Navar, L. G., Majid, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+inulin+in+tissues+using+anthrone.">Assay of inulin in tissues using anthrone.</a> Clinical Biochemistry. 8 (1), 67-70 (1975).</li><li>Shalmi, M., Lunau, H. E., Petersen, J. S., Bak, M., Christensen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+tritiated+inulin+for+determination+of+glomerular+filtration+rate.">Suitability of tritiated inulin for determination of glomerular filtration rate.</a> Americam Journal of Physiology. 260 (2 Pt 2), F283-F289 (1991).</li><li>Denton, K. M., Anderson, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+untrafiltration+in+rabbits+with+superficial+glomeruli.">Glomerular untrafiltration in rabbits with superficial glomeruli.</a> EUropean Journal of Physiology. 419 (3-4), 235-242 (1991).</li><li>Jobin, J., Bonjour, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+glomerular+filtration+rate+in+conscious+unrestrained+rats+with+inulin+infused+by+implanted+osmotic+pumps.">Measurement of glomerular filtration rate in conscious unrestrained rats with inulin infused by implanted osmotic pumps.</a> Americam Journal of Physiology. 248 (5 Pt 2), F734-F738 (1985).</li><li>Lorenz, J. N., Gruenstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+nonradioactive+method+for+evaluating+single-nephron+filtration+rate+using+FITC-inulin.">A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin.</a> Americam Journal of Physiology. 276 (1 Pt 2), F172-F177 (1999).</li><li>Qi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+determination+of+glomerular+filtration+rate+in+conscious+mice+using+FITC-inulin+clearance.">Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance.</a> Americam Journal of Physiology. 286 (3), F590-F596 (2004).</li><li>Bivona, B. J., Park, S., Harrison-Bernard, L. 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>

An appropriate marker for GFR measurement must meet four criteria: be freely filtered at the glomerulus, be unbound to plasma proteins, and neither be absorbed nor secreted in the nephron. Inulin is a fructose polymer which satisfies these criteria. As a result, the renal clearance of inulin is considered the gold standard for measuring GFR7. The demonstrated technique represents the traditional approach of determining the renal clearance of inulin using timed urine collections during a constant infusion of in...

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.

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	<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&#160;</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.&#160;</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&#160;</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&#160;</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&#160; Co</td>
			<td>105-420</td>
			<td></td>
		</tr>
	</tbody>
</table>
5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar&#160; 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.&#160; #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale&#160; 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&#160; 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&#160; Co 105-420

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.

The diuretic used in the lab demonstration was furosemide which very quickly inhibits the reabsorption of Na and K filtered by the kidney resulting in increased Na, K, and water excretion within minutes of drug administration. By its primary mechanism, furosemide should have minimal effects on GFR and the filtered load of Na and K, but will increase urine flow, and fractional excretion of Na and K.
The representative results in Table 3 show that in an anesthetized rat, the ave...

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

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.

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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