title: Fluorescence dilution technique for measurement of albumin reflection coefficient in isolated glomeruli.
abstract: This study describes a high-throughput fluorescence dilution technique to measure the albumin reflection coefficient (σAlb) of isolated glomeruli. Rats were injected with FITC-dextran 250 (75 mg/kg), and the glomeruli were isolated in a 6% BSA solution. Changes in the fluorescence of the glomerulus due to water influx in response to an imposed oncotic gradient was used to determine σAlb. Adjustment of the albumin concentration of the bath from 6 to 5, 4, 3, and 2% produced a 10, 25, 35, and 50% decrease in the fluorescence of the glomeruli. Pretreatment of glomeruli with protamine sulfate (2 mg/ml) or TGF-β1 (10 ng/ml) decreased σAlb from 1 to 0.54 and 0.48, respectively. Water and solute movement were modeled using Kedem-Katchalsky equations, and the measured responses closely fit the predicted behavior, indicating that loss of albumin by solvent drag or diffusion is negligible compared with the movement of water. We also found that σAlb was reduced by 17% in fawn hooded hypertensive rats, 33% in hypertensive Dahl salt-sensitive (SS) rats, 26% in streptozotocin-treated diabetic Dahl SS rats, and 21% in 6-mo old type II diabetic nephropathy rats relative to control Sprague-Dawley rats. The changes in glomerular permeability to albumin were correlated with the degree of proteinuria in these strains. These findings indicate that the fluorescence dilution technique can be used to measure σAlb in populations of isolated glomeruli and provides a means to assess the development of glomerular injury in hypertensive and diabetic models.
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Fan
authors.initials: F
authors.lastname: Fan
authors.affiliation: Department of Pharmacology, Lake Erie College of Osteopathic Medicine, Erie, Pennsylvania;
authors.forename: Chun Cheng Andy
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Nephrology, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina;
authors.forename: Carlos M N
authors.initials: CM
authors.lastname: Schreck
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi; rroman@umc.edu.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Roman
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Jan M
authors.initials: JM
authors.lastname: Williams
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Takashi
authors.initials: T
authors.lastname: Hirata
authors.affiliation: Department of Nephrology, Internal Medicine, Kansas City Veterans Affairs Medical Center, Kansas City, Missouri; and.
authors.forename: Mukut
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Department of Molecular and Integrative Physiology, Internal Medicine, University of Michigan, Ann Arbor, Michigan.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Beard
authors.affiliation: Department of Nephrology, Internal Medicine, Kansas City Veterans Affairs Medical Center, Kansas City, Missouri; and.
authors.forename: Virginia J
authors.initials: VJ
authors.lastname: Savin
authors.affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roman
mesh_terms: D000418:Albumins / Q000032:analysis*; D000818:Animals; D003921:Diabetes Mellitus, Experimental; D003928:Diabetic Nephropathies / Q000652:urine*; D005453:Fluorescence; D007201:Indicator Dilution Techniques; D007678:Kidney Glomerulus / Q000378:metabolism / Q000503:physiopathology*; D008810:Mice, Inbred C57BL; D010539:Permeability / Q000187:drug effects; D011507:Proteinuria / Q000139:chemically induced; D020303:Rats, Inbred Dahl; D017207:Rats, Sprague-Dawley; D013311:Streptozocin
chemical_list: D000418:Albumins; D013311:Streptozocin
keywords: glomerulus; kidney; proteinuria; renal disease; renal hemodynamics