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.