A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin

Abstract

The determination of inulin concentration in nanoliter fluid samples is fundamental to micropuncture investigations of renal function, and this is generally accomplished through the use of radioisotopes. We report here a simple and reliable alternative to the use of radioisotopes that employs FITC-labeled inulin. Samples containing FITC-inulin are stored between oil columns in constant-bore microcapillary tubes, which are then used as cuvettes to determine fluorescence on a microscope fluorometer. Standard curves were generated and found to be linear, with correlation coefficients ( R) exceeding 0.99 in every case. Although the fluorescence of FITC-inulin was found to be pH dependent, the pH and fluorescence of each 20- to 40-nl sample could be normalized by the addition of 1 nl of 0.5 M HEPES at pH 7.5. In mice prepared for standard micropuncture, simultaneous measurements of tubular fluid-to-plasma ratios (TF/P) using FITC-inulin and [125I]iothalamate were highly correlated (slope = 0.95, y-intercept = 0.01, R = 0.942), as were whole kidney measurements of glomerular filtration rate (GFR) (slope = 1.25, y-intercept = −53.5 μl/min, R = 0.99). Micropuncture determinations of late-proximal samples from mice before and after treatment with acetazolamide showed expected changes: TF/P of FITC-inulin decreased from 1.89 ± 0.07 to 1.48 ± 0.10; single-nephron GFR (SNGFR) decreased from 9.64 ± 1.1 to 6.65 ± 1.0 nl/min; and fractional fluid reabsorption decreased from 45.3 ± 1.9 to 26.8 ± 5.2%. Measurements of TF/P of FITC-inulin, volume, and SNGFR using this technique were stable for at least 2 wk when samples were stored in the dark at 4°C. These data demonstrate that this simple method for determining inulin clearance represents a viable and accurate alternative to radioactive methods. This approach has the added benefits of being relatively inexpensive and leaving the micropuncture sample intact.

The Isolated Perfused Rat Kidney: A Technical Update

“…In both cases, the increased absorbance would lead to an underestimation of GFR. Consequently, these findings support the use of alternative methods for inulin quantitation such as fluorescently tagged inulin [4].…”

“…Our data suggests that caution needs to be exercised when using either high glucose or dextran in combination with inulin as a marker for GFR. Under these circumstances, the use of alternative methods of inulin quantitation such as fluorescently tagged inulin is preferable [4]. These considerations will be of particular relevance to researchers utilizing the isolated perfused kidney as a screening tool to measure renal clearance, and drug metabolism as well as investigations into diabetic nephropathy.…”

Protective effect of renal denervation on normotensive endotoxemia-induced acute renal failure in mice

“…An intravenous maintenance infusion of 2.25% BSA in normal saline at a rate of 0.25 l ⅐ g Ϫ1 ⅐ min Ϫ1 was started 1 h before experimentation. FITC-inulin (0.75%; Sigma, St Louis, MO) was added to the infusion solution for the determination of GFR, as described by Lorenz and Gruenstein (10). A bladder catheter (PE-10) was used to collect urine.…”

Thromboxane Receptor Mediates Renal Vasoconstriction and Contributes to Acute Renal Failure in Endotoxemic Mice

“…The right femoral artery was cannulated with a tapered PE-100 catheter connected to a pressure transducer (Statham P23 DB) for continuous monitoring of MAP. FITC-inulin (0.25%; Sigma, St. Louis, MO) was added to BSA infusion for determination of GFR by clearance methodology (22). A PE-50 catheter was introduced into the bladder to collect urine.…”