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><tbody><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>FITC-inulin</td><td>Sigma-Aldrich</td><td>F3272</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H7706</td><td></td></tr><tr><td>Isoflurane</td><td>Webster Veterinary</td><td>78366551</td><td></td></tr><tr><td>Dialysis membrane (MWCO 1000)</td><td>Spectrum Laboratories</td><td>132636</td><td></td></tr><tr><td>Membrane closure</td><td>Spectrum Laboratories</td><td>132736</td><td></td></tr><tr><td>80 &amp;mu;l Hematocrit capillaries</td><td>Drummond Scientific</td><td>22362566</td><td></td></tr><tr><td>Hematocrit centrifuge</td><td>IEC</td><td>Micro-MB</td><td></td></tr><tr><td>10 &amp;mu;l Na&lt;sup&gt;+&lt;/sup&gt;-Heparin minicaps</td><td>Hirschmann</td><td>9000210</td><td></td></tr><tr><td>Syringe (100 &amp;mu;l)</td><td>Hamilton</td><td>80601</td><td></td></tr><tr><td>Fluorospectrometer</td><td>Thermo-Fisher Scientific</td><td>NanoDrop 3300</td><td></td></tr><tr><td>Filter (0.22 &amp;mu;m)</td><td>Spectrum Laboratories</td><td>880-26464-UE</td><td></td></tr><tr><td>Sealant</td><td>Ch&amp;#226;-seal</td><td>510</td><td></td></tr><tr><td>Needles (G26 &amp;frac12; and G30 &amp;frac12;)</td><td>Becton Dickinson</td><td>305111 &amp;amp; 305106</td><td></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.

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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 ...

a-high-throughput-method-for-measurement-glomerular-filtration-rate

Nanyang Technological University

Research

JoVE Journal

Biology

43.2K Views.  University of California, San Diego. 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.

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

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.

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.

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.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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 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.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

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 ...

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.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...