Inulin clearance > Protocol Section
Preparation of 5% FITC-inulin solution:
- 5% FITC-inulin is prepared by dissolving FITC-inulin in two ml of 0.9% NaCl -- facilitated by heating the solution in boiling water.
- To remove residual FITC not bound to inulin, the solution is filled into a 1000 Daltons cut-off dialysis membrane (Spectra/Pro 6, Spectrum Laboratories Inc., Rancho Dominguez, CA).
- Put the dialysis membrane filled with FITC-inulin into 1000 ml 0.9% NaCl for 24 hours at room temperature.
- Prior to use, this dialyzed solution is sterilized by filtration through a 0.22 μm filter (e,g, Costar).
Intravenous injection and blood collection:
- Mice are anesthetized using Isoflurane.
- 5% FITC-inulin (3.74μl /g body weight) is injected retroorbitally under anesthesia within 10 seconds.
- After fully regaining consciousness, the mouse is restrained inside a 50-mL Falcon tube with an air-hole drilled in the tip.
- The inner thigh was shaven and wiped with 75% ethanol, revealing the saphenous vein. Approximately 20 μl blood is collected in a heparinized capillary tube (Fisher Scientific) by puncture of the vein using a sterile 23 gauge syringe needle (3). On average, this yields 10 μl of plasma following centrifugation (4,000 RPM, 10 min).
- Blood is sampled via the saphenous vein at 3, 7, 10, 15, 35, 55, 75 minutes post injection of FITC-inulin.
Determination of fluorescence of the sampled plasma:
- Since pH significantly affects FITC fluorescence value, each plasma sample is buffered to pH 7.4, by mixing 10 μl of plasma with 40 μl of 500 mM HEPES (pH 7.4).
- The titrated samples are then loaded onto a 96-well plate, 50μl sample/well. Fluorescence is determined using a Fluoroscan Ascent FL (Labsystems, FIN-00811 Helsinki, Finland), with 485 nm excitation, and read at 538 nm emission.
Calculation of GFR:
A two-compartment clearance model is employed for the calculation of GFR (1, 2). In the two-compartment model, as depicted in Fig 1, the initial, rapid decay phase represents primarily redistribution of the tracer from the intravascular compartment to the extracellular fluid compartment, while systemic elimination constitute a minor part. During the later, slower decay phase, systemic clearance of the tracer from the plasma predominates. At any given time (tx), the plasma concentration of the tracer (Y) equals to Ae-αtx+Be-βtx +Plateau (http://curvefit.com/id205.htm).
The parameters of above equation could be calculated using a non-linear regression curve-fitting program (GraphPad Prism, GraphPad Software, Inc., San Diego, CA.) (see below). GFR was calculated using the equation: GFR= I/(A/α + B/β), where I is the amount of FITC-inulin delivered by the bolus injection; A (Span1) and B (Span2) are the y -intercept values of the two decay rates, and α and β are the decay constants for the distribution and elimination phases, respectively.
Use of Prism:
- Open Prism program and click “New table”.
- Input time point into “X Values” column, and the corresponding fluorescence data into “ Y” column.
- Click “ Analysis” and choose “ Nonlinear regression (curve fit)”
- Click “ OK”, and choose “ Two phase exponential decay”
- Set “Plateau” zero.
- The parameters of the fluorescence decay curve are shown in a spreadsheet. SPAN1 and SPAN2 are Y intercepts , and K1 and K2 are constants of the distribution and clearance phases, respectively.
Publications for Inulin clearance (3)
Qi Z, Whitt I, Mehta A, Jin J, Zhao M, Harris RC, Fogo AB, Breyer MD. Serial determination of glomerular filtration rate in conscious mice using
FITC-inulin clearance. Am J Physiol Renal Physiol (2004) 286:F590-6
View abstract View in PubMed
Two nonradioactive methods for determining glomerular filtration rate (GFR) in conscious mice using FITC-labeled inulin (FITC-inulin) were evaluated. The first method measured GFR using clearance kinetics of plasma FITC-inulin after a single bolus injection. Based on a two-compartment model, estimated GFR was 236.69 +/- 16.55 and 140.20 +/22.27 microl/min in male and female C57BL/6J mice, respectively. Total or (5/6) nephrectomy reduced inulin clearance to 0 or 32.80 +/9.32 microl/min, respectively. Conversely, diabetes mellitus induced by streptozotocin was associated with increased GFR. The other approach measured urinary inulin clearance using intraperitoneal microosmotic pumps to deliver FITC-inulin and metabolic cages to collect timed urine samples. This approach yielded similar GFR values of 211.11 +/26.56 and 157.36 +/20.02 microl/min in male and female mice, respectively. These studies demonstrate the feasibility of repeated nonisotopic measurement of inulin clearance in conscious mice.
A method is described for blood collection from the lateral saphenous vein. This enables rapid sampling, which if necessary can be repeated from the same site without a need for new puncture wounds. The method is a humane and practical alternative to cardiac and retro-orbital puncture, in species where venepuncture has traditionally been regarded as problematic.
Sturgeon C, Sam AD 2nd, Law WR. Rapid determination of glomerular filtration rate by single-bolus inulin: a
comparison of estimation analyses. J Appl Physiol (1985) (1998) 84:2154-62
View abstract View in PubMed
Rapid measurement of glomerular filtration rate (GFR) by an inulin single-bolus technique would be useful, but its accuracy has been questioned. We hypothesized that reported inaccuracies reflect the use of inappropriate mathematical models. GFR was measured in 14 intact and 5 unilaterally nephrectomized conscious male Sprague-Dawley rats (mean weight 368 +/12 g) by both single-bolus (25 mg/kg) and constant-infusion techniques (0.693 mg . kg-1 . min-1). The temporal decline in plasma inulin concentration was analyzed through biexponential curve fitting, which accounted for renal inulin loss before complete vascular and interstitial mixing. We compared our mathematical model based on empirical rationale with those of other investigators whose studies suggest inaccuracy of single-bolus methods. Our mathematical model yielded GFR values by single bolus that agreed with those obtained by constant infusion [slope = 0.94 +/0.16 (SE); y intercept = 0.23 +/0.64; r = 0.82]. In comparison to the data obtained by constant inulin infusion, this method yielded a very small bias of -0.0041 +/0.19 ml/min. Two previously reported models yielded unsatisfactory values (slope = 1. 46 +/0.34, y intercept = 0.47 +/1.5, r = 0.72; and slope = 0.17 +/1.26, y intercept = 17.15 +/5.14, r = 0.03). The biases obtained by using these methods were -2.21 +/0.42 and -13.90 +/1. 44 ml/min, respectively. The data indicate that when appropriate mathematical models are used, inulin clearance after single-bolus delivery can be used to measure GFR equivalent to that obtained by constant infusion of inulin. Attempts to use methods of analysis for simplicity or expediency can result in unacceptable measurements relative to the clinical range of values seen.
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Last updated on 2013-11-06 Moderated by Jimmy Hao