Name: a high-throughput method for measurement of glomerular filtration rate in conscious mice
Uploaded: 2013-05-10T12:00:00
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Description: Watch this Scientific Journal Video about A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice at JoVE.com

I thank Dr. Jessica Dominguez Rieg for critical reading of this manuscript. This work was supported by the American Heart Association (Scientist Development Grant 10SDG2610034), a Carl W. Gottschalk Research Grant of the American Society of Nephrology, the National Institute of Diabetes and Digestive and Kidney Diseases O'Brien Center for Acute Kidney Injury Research (P30DK079337), and the Department of Veterans Affairs.

1. Solutions
<ol>
	<li>0.85% NaCl: 8.5 g NaCl/1 l ddH2O (8.5 g/L).</li>
	<li>0.5 mol/L HEPES pH 7.4 (dissolve 59.6 g of HEPES in 0.5 L of ddH20 and adjust pH to 7.4 using 10 N NaOH).</li>
</ol>
2. Preparation of FITC-inulin Injection Solution
<ol>
	<li>Weigh FITC-inulin to prepare a 5% solution and dissolve in 0.85% NaCl (usually 100 mg/2 ml 0.85% NaCl) by heating to 90 &deg;C until completely dissolved.</li>
	<li>Weigh dissolved FITC-inulin solution and note weight.</li>
	<li>Cut a 20 cm piece of dialysis membrane (molecular weight cut-off: 1,000) and put in ddH2O for 30 min to remove residual NaN3 from the membrane and flush a few times afterwards.</li>
	<li>Fill dissolved FITC-inulin into dialysis membrane and seal properly with closures.</li>
	<li>Weigh entire dialysis membrane.</li>
	<li>Put the dialysis membrane in 1 L 0.85% NaCl solution and stir slowly light-protected for 24 hr at room temperature.</li>
	<li>Weigh entire dialysis membrane again and calculate the new concentration (water will osmotically move into the membrane and unbound FITC, or bound FITC-inulin &lt;1,000 molecular weight will move out of the membrane; thus, the concentration of FITC-inulin will decrease substantially).</li>
	<li>The new FITC-inulin concentration (c) is calculated using the following formula: c = n/V, where n = initial FITC-inulin amount, V = new volume (difference in weight of dialysis tubing before and after dialysis plus volume of initial FITC-Inulin solution).</li>
	<li>Prior to use sterilize dialyzed FITC-inulin solution by filtration through a 0.22 &mu;m syringe filter.</li>
	<li>Keep FITC-inulin protected from light (aluminum foil) at 4 &deg;C. Dialyzed and sterilized FITC-inulin can be used for up to 2 weeks. Participated FITC-inulin can be dissolved by re-heating at 90 &deg;C for a few minutes.</li>
</ol>
Note: if using FITC-sinistrin 16-18, which does not require dialysis, all steps required for dialysis can be skipped.
3. Injection and Blood Collection
<ol>
	<li>Take body weight (bw) of the mice before the experiment.</li>
	<li>Anesthetize mice briefly with isoflurane.</li>
	<li>For high-throughput of 6 mice please follow protocol provided in Figure 3.</li>
	<li>Inject 2 &mu;l/g bw of dialyzed FITC-inulin into the retroorbital plexus 24 by using a G30 &frac12; in needle (remove air bubbles from 100 &mu;l Hamilton syringe by first using a G26 &frac12; in needle and then switching to a G30 &frac12; in needle for injection).</li>
	<li>Cut 1 mm of mouse tail with scissors once and collect blood at 3, 5, 7, 10, 15, 35, 56 and 75 min after injection in Na-Heparin minicapillaries. Seal minicapillaries after blood collection with Ch&acirc;-seal.</li>
	<li>Keep samples protected from light.</li>
	<li>Put sealed minicapillaries inside hematocrit capillaries and centrifuge for 5 min.</li>
	<li>Break minicapillaries by using a diamond cutter and transfer entire plasma by pipetting into a 0.2 ml tube.</li>
	<li>Make a 1:10 dilution by using 2 &mu;l of plasma and 18 &mu;l 0.5 mol/L HEPES (pH 7.4) in new 0.2 ml tube (samples could be stored overnight at 4 &deg;C for fluorescence measurements).</li>
	<li>Measure 2 &mu;l of diluted sample with NanoDrop 3300 in duplicates. The description and usage of the instrument is described here 5.</li>
</ol>
4. Preparation of Standards
<ol>
	<li>Collect blood from mice of the same background strain in heparin coated hematocrit capillaries, spin down and dilute 1:10 with 0.5 mol/L HEPES (pH 7.4); usually 80 &mu;l plasma + 720 &mu;l HEPES.</li>
	<li>Dilute FITC-inulin injection solution with diluted mouse plasma to get the following standard dilutions: 
	1:10: 10 &mu;l undiluted FITC-inulin solution + 90 &mu;l diluted mouse plasma 
	1:100: 10 &mu;l (1:10) solution + 90 &mu;l diluted mouse plasma 
	1:1,000: 10 &mu;l (1:100) solution + 90 &mu;l diluted mouse plasma 
	1:2,000: 30 &mu;l (1:1) solution + 30 &mu;l diluted mouse plasma 
	1:10,000: 10 &mu;l (1:5) solution + 40 &mu;l diluted mouse plasma 
	1:20,000: 20 &mu;l (1:1) solution + 20 &mu;l diluted mouse plasma 
	1:100,000: 10 &mu;l (1:5) solution + 40 &mu;l diluted mouse plasma 
	The concentration of the standards will depend on the dialysis and will always be different for each preparation of FITC-inulin. It is necessary to enter the concentration for the standard curve new every time.</li>
	<li>Analyze data to calculate GFR with appropriate software (e.g. GraphPad Prism) by using a two-phase exponential decay function as described in here 15, 20 and shown in Representative Results Section.</li>
</ol>

<ol>
	<li>Bauer, J. H., Brooks, C. S., Burch, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+appraisal+of+creatinine+clearance+as+a+measurement+of+glomerular+filtration+rate.">Clinical appraisal of creatinine clearance as a measurement of glomerular filtration rate.</a> Am. J. Kidney Dis. 2 (3), 337-346 (1982).</li><li>Carrie, B. J., Golbetz, H. V., Michaels, A. S., Myers, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatinine:+an+inadequate+filtration+marker+in+glomerular+diseases.">Creatinine: an inadequate filtration marker in glomerular diseases.</a> Am. J. Med. 69 (2), 177-182 (1980).</li><li>Chasis, H., Ranges, H. A., Goldring, W., 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+Control+of+Renal+Blood+Flow+and+Glomerular+Filtration+in+Normal+Man.">The Control of Renal Blood Flow and Glomerular Filtration in Normal Man.</a> J. Clin. Invest. 17 (5), 683-697 (1938).</li><li>Darling, I. M., Morris, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+"true"+creatinine+clearance+in+rats+reveals+extensive+renal+secretion.">Evaluation of "true" creatinine clearance in rats reveals extensive renal secretion.</a> Pharm. Res. 8 (10), 1318-1322 (1991).</li><li>Dunn, S. R., Qi, Z., Bottinger, E. P., Breyer, M. D., Sharma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endogenous+creatinine+clearance+as+a+measure+of+renal+function+in+mice.">Utility of endogenous creatinine clearance as a measure of renal function in mice.</a> Kidney Int. 65 (5), 1959-1967 (2004).</li><li>Eisner, C., Faulhaber-Walter, R., 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=Major+contribution+of+tubular+secretion+to+creatinine+clearance+in+mice.">Major contribution of tubular secretion to creatinine clearance in mice.</a> Kidney Int. 77 (6), 519-526 (2010).</li><li>Faulhaber-Walter, R., Chen, L., 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=Lack+of+A1+adenosine+receptors+augments+diabetic+hyperfiltration+and+glomerular+injury.">Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury.</a> J. Am. Soc. Nephrol. 19 (4), 722-730 (2008).</li><li>FINGL, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+excretion+of+creatinine+in+the+rat.">Tubular excretion of creatinine in the rat.</a> Am. J. Physiol. 169 (2), 357-362 (1952).</li><li>Keppler, A., Gretz, N., 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=Plasma+creatinine+determination+in+mice+and+rats:+an+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: an enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71 (1), 74-78 (2007).</li><li>Levey, A. S., Perrone, R. D., Madias, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+creatinine+and+renal+function.">Serum creatinine and renal function.</a> Annu. Rev. Med. 39, 465-490 (1988).</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> Am. J. Physiol. 276, 172-177 (1999).</li><li>Meyer, M. H., Meyer, R. A., Gray, R. W., Irwin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picric+acid+methods+greatly+overestimate+serum+creatinine+in+mice:+more+accurate+results+with+high-performance+liquid+chromatography.">Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography.</a> Anal. Biochem. 144 (1), 285-290 (1985).</li><li>Qi, Z., Breyer, M. D. <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+mice.">Measurement of glomerular filtration rate in conscious mice.</a> Methods Mol. Biol. 466, 61-72 (2009).</li><li>Qi, Z., Whitt, I., 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> Am. J. Physiol. Renal Physiol. 286 (3), F590-F596 (2004).</li><li>Schock-Kusch, D., Sadick, M., 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=Transcutaneous+measurement+of+glomerular+filtration+rate+using+FITC-sinistrin+in+rats.">Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats.</a> Nephrol. Dial Transplant. 24 (10), 2997-3001 (2009).</li><li>Schock-Kusch, D., Xie, Q., 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=Transcutaneous+assessment+of+renal+function+in+conscious+rats+with+a+device+for+measuring+FITC-sinistrin+disappearance+curves.">Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.</a> Kidney Int. 79 (11), 1254-1258 (2011).</li><li>Schreiber, A., Shulhevich, Y., 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=Transcutaneous+measurement+of+renal+function+in+conscious+mice.">Transcutaneous measurement of renal function in conscious mice.</a> Am. J. Physiol. Renal Physiol. 303 (5), F783-F788 (2012).</li><li>Shemesh, O., Golbetz, H., Kriss, J. P., Myers, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+creatinine+as+a+filtration+marker+in+glomerulopathic+patients.">Limitations of creatinine as a filtration marker in glomerulopathic patients.</a> Kidney Int. 28 (5), 830-838 (1985).</li><li>Sturgeon, C., Sam, A. D., Law, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+glomerular+filtration+rate+by+single-bolus+inulin:+a+comparison+of+estimation+analyses.">Rapid determination of glomerular filtration rate by single-bolus inulin: a comparison of estimation analyses.</a> J. Appl. Physiol. 84 (6), 2154-2162 (1998).</li><li>Vallon, V., Blantz, R. C., Thomson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+hyperfiltration+and+the+salt+paradox+in+early+[corrected]+type+1+diabetes+mellitus:+a+tubulo-centric+view.">Glomerular hyperfiltration and the salt paradox in early [corrected] type 1 diabetes mellitus: a tubulo-centric view.</a> J. Am. Soc. Nephrol. 14 (2), 530-537 (2003).</li><li>Vallon, V., Eraly, S. A., 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=A+role+for+the+organic+anion+transporter+OAT3+in+renal+creatinine+secretion+in+mice.">A role for the organic anion transporter OAT3 in renal creatinine secretion in mice.</a> Am. J. Physiol. Renal Physiol. 302 (10), F1293-F1299 (2012).</li><li>Vallon, V., Schroth, J., 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=Adenosine+A(1)+receptors+determine+glomerular+hyperfiltration+and+the+salt+paradox+in+early+streptozotocin+diabetes+mellitus.">Adenosine A(1) receptors determine glomerular hyperfiltration and the salt paradox in early streptozotocin diabetes mellitus.</a> Nephron. Physiol. 111 (1), 30-38 (2009).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Anim. (NY. 40 (5), 155-160 (2011).</li></ol>

The author received funding to cover the costs of this publication by Thermo Fisher Scientific.

The present study describes a technique for determination of GFR in conscious mice by single-bolus injection and analysis of fluorescence in 8 blood samples by two-phase exponential decay. A GFR measurement in 1 mouse takes 75 min. By using a special flow-chart, it is possible to measure the GFR of 6 mice in 130 min. It is feasible to repeat this flow-chart protocol up to 4 times per day and measure GFR in 24 mice. We modified this method so that it is possible to collect blood from a small tail snip rather than tail vein puncture, which is quicker and easier for investigators to perform compared to tail vein puncture. By using 10 &mu;l hematocrit capillaries and a 1:10 dilution, it is possible to reduce the total amount of blood needed to &lt;100 &mu;l. Finally, fluorescence is measured using a NanoDrop 3300 fluorospectrometer, which allows determination of fluorescence in 1-2 &mu;l of diluted sample. This method will give the interested investigator a true GFR value. In contrast, creatinine measurements in mice have several weaknesses including "creatinine blind range" and difficulties related to specific and sensitive determination. The "creatinine blind range" limits its function as a marker for GFR because serum creatinine remains in the normal range until 50% of renal function is lost 19. Since several investigators utilize genetically modified mice in their research to study renal physiology and pathophysiology, this bears the following problem: assuming a knockout mouse of gene X would have a significantly lower GFR (by 40%) compared to a wild-type mouse, a simple screening by comparing plasma creatinine would not detect this difference. The need for a good accurate method of GFR determination becomes more critical the smaller the differences in GFR become.
Mice have high concentrations of non-creatinine chromagens in their plasma and urine that interfere with commercially available creatinine assays. As a consequence, assays which are based on the alkaline picrate Jaff&eacute; reaction overestimate creatinine concentration in plasma compared with high performance liquid chromatography (HPLC) determination 6, 13. Another limitation is that serum creatinine is below detection limits of several commercially available assays compared to HPLC determination. However, by using enzymatic reactions investigators were able to determine plasma creatinine in mice 10, 22.
GFR in a similar range were found in conscious mice using a comparable single-bolus injection method as well as urinary FITC-inulin clearance 14. In contrast to their protocol, our protocol using tail snip does not require special precautions to preserve the vessel integrity compared to tail vein or saphenous vein puncture. For the urinary/plasma based FITC-inulin clearance it is necessary to surgically implant two osmotic minipumps. Two pumps are necessary to achieve high enough steady-state plasma FITC-inulin concentrations which are clearly distinguishable from the background. This also requires the use of metabolic cages for a complete and timed urine collection. The cages need to be rinsed to account for a loss of 25-37 % of FITC-inulin, which otherwise occurs without rinsing.
The pitfalls of using this FITC-inulin method are limited. Investigators should be aware of the following: (i) assure that there are no air bubbles in the syringe used for injection of the FITC-solution, (ii) complete retrobulbar injection must be achieved because the amount injected determines the calculated GFR, and (iii) mice will most likely urinate during the 75 min GFR experiment, so avoid contaminating blood samples with urine, which will have a very high FITC-inulin concentration as a consequence of the kidneys concentration ability.
General advantages of this FITC-inulin method include the following: (i) it is a non-radioactive technique, (ii) it is possible to repeat measurements multiple times in the same mouse, (iii) the assay can be performed in conscious mice, (iv) no urine collection is required, and (v) a high-throughput option can be utilized. Disadvantages for the single bolus injection include the need for dialysis of FITC-inulin, which can be overcome by using a better FITC-marker, FITC-sinistrin, which is available now and can be measured transcutaneously using a special miniaturized device 18. Even though GFR is measured, it is not possible to measure fractional excretions of fluid or ions with this method.
Taken together, this method provides the feasibility of a high-throughput method for measuring GFR in conscious mice. Thus, this technique might be of interest for other investigators who are interested in determining renal function in mice.

The rate of formation of plasma ultrafiltrate at the glomerulus has become the basis for evaluating renal function (glomerular filtration rate, GFR). The ideal marker to determine GFR should be freely filtered, neither secreted or reabsorbed and not metabolized. Such a behavior was found to be true for inulin, and inulin clearance has become a gold standard for determination of GFR 3. Whereas the clearance of creatinine is extensively used as a measure of GFR in humans and animals, creatinine does not fulfill the requirements of an ideal GFR marker. Around 10 - 40% of the creatinine clearance in humans is the consequence of active tubular secretion 1, 11, and renal secretion of creatinine is also prominent in mice and rats 4, 7, 9, 22. Renal dysfunction is known to increase the tubular secretion of creatinine which limits its value as a marker of GFR 1, 2. We have previously shown that organic anion transporter isoform 3 (OAT3) contributes to murine renal creatinine secretion 22. However, inulin clearance typically requires the use of radioactive labeled inulin (3H or 14C). The radioactive determination of inulin can be used in both anesthetized and conscious mice, but harbors the disadvantages of numerous safety regulations and strict handling issues inherent with the use of radioactive isotopes. Along those lines, surgical clearance preparations for determination of GFR are time consuming as well as terminal in nature. In 1999 Lorenz et al. 12 introduced FITC-inulin as a marker of single nephron GFR in anesthetized mice. Five years later Qi et al. 15 determined whole kidney GFR in conscious mice by using FITC-inulin. Alternative protocols are now using FITC-inulin as an exogenous marker to measure GFR in conscious and anesthetized mice. These protocols retain the sensitivity of radioactive inulin by using fluorescence detection and circumventing the disadvantages of working with a radioactive labeled marker. The FITC-inulin assay was modified by others 7, 8 and us 22, 23 to take advantage of the microvolume capability of the NanoDrop 3300 fluorospectrometer. This allows reducing the total sample volume needed to &lt;100 &mu;l blood per GFR measurement.
With the method and high throughput protocol provided in this publication, we demonstrate the feasibility of measuring GFR in up to 24 conscious mice per day. This technique could be useful to other investigators studying murine GFR and we hope it will replace less accurate methods as indices for kidney function, such as creatinine and blood urea nitrogen.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FITC-inulin</td>
			<td>Sigma-Aldrich</td>
			<td>F3272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7706</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Webster Veterinary</td>
			<td>78366551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dialysis membrane (MWCO 1000)</td>
			<td>Spectrum Laboratories</td>
			<td>132636</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Membrane closure</td>
			<td>Spectrum Laboratories</td>
			<td>132736</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>80 &mu;l Hematocrit capillaries</td>
			<td>Drummond Scientific</td>
			<td>22362566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hematocrit centrifuge</td>
			<td>IEC</td>
			<td>Micro-MB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 &mu;l Na+-Heparin minicaps</td>
			<td>Hirschmann</td>
			<td>9000210</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe (100 &mu;l)</td>
			<td>Hamilton</td>
			<td>80601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorospectrometer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>NanoDrop 3300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter (0.22 &mu;m)</td>
			<td>Spectrum Laboratories</td>
			<td>880-26464-UE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sealant</td>
			<td>Ch&acirc;-seal</td>
			<td>510</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needles (G26 &frac12; and G30 &frac12;)</td>
			<td>Becton Dickinson</td>
			<td>305111 &amp; 305106</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

GFR will be calculated using the following formula:
GFR = n/(A/K1 + B/K2), where
n = injected amount (n = c&bull;V, where c = FITC-inulin concentration, V = injected volume) 
A = Span1, y-intercept of elimination 
K1= decay constant for elimination 
B = Span2, y-intercept of distribution 
K2 = decay constant for distribution
Note: A, K1, B and K2 are abbreviations given by GraphPad Prism, every software capable of analyzing two-phase exponential decay can be used, however, other scientific software might use different abbreviations.
By employing a two-phase exponential decay curve fit, which is based on plasma FITC-inulin concentrations, a curve comparable to the one shown in Figure 1 should be obtained. Calculating GFR by the above shown formula (and as described in here 20) allows to detect impairment of kidney function 15 or glomerular hyperfiltration 8, 23 as found in mice with type 1 diabetes mellitus (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/50330/50330fig1.jpg" /> 
 Figure 1. To measure GFR in conscious mice, plasma kinetics of FITC-inulin following a single-dose intravenous injection is used. For calculation of GFR, a two-compartment model is employed. In the two-compartment model, the initial rapid decay phase represents redistribution of FITC-inulin from the intravascular compartment to the extracellular fluid. The later phase, with slower decay in FITC-inulin concentration, predominantly reflects systemic clearance from the plasma.
<img alt="Figure 2" src="/files/ftp_upload/50330/50330fig2.jpg" /> 
 Figure 2. Detection of diabetic hyperfiltration in wild-type mice. Type I diabetes mellitus was induced by intraperitoneal application of streptozotocin (STZ, 50 mg/kg for 5 consecutive days). GFR was determined before (basal) and 5 weeks after induction of diabetes. Due to tubuloglomerular feedback, a primary increase in proximal reabsorption causes early diabetic hyperfiltration 21 a finding that is reproducible in wild-type mice (n=10, p&lt;0.01 vs. basal same mouse).
<img alt="Figure 3" src="/files/ftp_upload/50330/50330fig3.jpg"/> 
 Figure 3. Flow-chart for measurement of GFR in a set of 6 mice. To measure whole kidney GFR by the single bolus injection method, 8 blood samples must be collected at 3, 5, 7, 10, 15, 35, 56 and 75 min after FITC-inulin injection. Numbers indicate time points. Numbers in brackets indicate sample number. Time points left of the vertical red line indicate injection time points (at 0, 11, 22, 33, 44 and 55 min). Each mouse is marked with a different color. To get sequential blood collections follow the arrows. Grey boxes indicate when the next mouse must be prepared for isoflurane anesthesia prior to injection. Using this protocol the minimum time between 2 blood collections from different mice is 1 min.

Watch this Scientific Journal Video about A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice at JoVE.com

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

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.

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

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Research

JoVE Journal

Biology

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

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 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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...