title: 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 microliter/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 degreesC. 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.
authors.affiliation: Department of Molecular and Cellular Physiology, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: Lorenz
authors.forename: E
authors.initials: E
authors.lastname: Gruenstein
mesh_terms: D000042:Absorption / Q000187:drug effects; D000086:Acetazolamide / Q000494:pharmacology; D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005919:Glomerular Filtration Rate / Q000502:physiology*; D007444:Inulin / Q000378:metabolism; D007483:Iothalamic Acid / Q000378:metabolism; D008297:Male; D051379:Mice; D009399:Nephrons / Q000378:metabolism*; D009994:Osmolar Concentration; D011677:Punctures
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D005456:Fluorescent Dyes; D007483:Iothalamic Acid; D007444:Inulin; D016650:Fluorescein-5-isothiocyanate; D000086:Acetazolamide