A simple, non-radioactive method for evaluating single nephron filtration rate and fluid reabsorption using fitc-inulin. Lorenz, John N., and Eric Gruenstein . Departments of Molecular and Cellular Physiology 1, and Molecular Genetics, Biochemistry and Microbiology 2 University of Cincinnati College of Medicine, Cincinnati, OH 45267
APStracts 5:0168F, 1998.
The determination of inulin concentration in nanoliter fluid samples is fundamental to micropuncture investigations of renal function, and is generally accomplished through the use of radioisotopes. We report here a simple and reliable alternative to the use of radioisotopes which 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 20-40 nl sample could be normalized by the addition of 1nl of 0.5 M Hepes at pH 7.5. In mice prepared for standard micropuncture, simultaneous measurements of tubular fluid/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 GFR (slope = 1.25, y-intercept = -53.5, R = 0.99). Micropuncture determinations of late-proximal samples from mice before and after treatment with acetazolamide showed expected changes: TF/PFITC-inulin decreased from 1.89+/-0.07 to 1.48+/-0.10; SNGFR decreased from 9.64+/-1.1 to 6.65 +/-1.0 nl/min; fractional fluid reabsorption decreased from 45.3+/ -1.9 to 26.8+/-5.2%. Measurements of TF/PFITC-inulin, volume and SNGFR using this technique were stable for at least two weeks 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. 

Received 1 June 1998; accepted in final form 17 September 1998.
APS Manuscript Number F136-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 20 October 1998