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Departments of 1 Molecular and Cellular Physiology, and 2 Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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.
kidney; renal micropuncture; glomerular filtration rate; fluorescein; nanoliter; fluid reabsorption; proximal tubule; mouse
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