Dilutional fluorescent method to measure glomerular albumin reflection coefficient (dσAlb) in vitro

Abstract

This study describes a new high throughput method (7‐15 glomeruli/experiment, 3 experiments/hour) to measure dσAlb. Adult SD rats received FITC‐Dextran 250 ( 75 mg/Kg. iv) and 5 minutes later the kidneys were harvested and glomeruli isolated using a sieving technique in 6% BSA. They were studied in a small volume (50 μl) fast exchange chamber on an inverted fluorescent microscope equipped with InCytIm 1 imaging system using a 10X (0.3 NA) objective. Changes in the fluorescent signal in the glomerular capillaries due to water movement in response to an imposed oncotic gradient were used to determine dóAlb. Changing the bath from 6 to 5, 4, 3, 2 % albumin produced a linear 12, 26, 37 and 52% fall in the concentration of FITC‐Dextran250 in the glomerulus ( r2=0.95). Administration of protamine sulfate ( 2 mg/ml) or TGF‐β1 (10 ng/ml) to increase the glomerular permeabilty to albumin, decreased dσAlb from 1 to 0.6 and from 1 to 0.67 , respectively. Fluorescence increased 10 % in glomeruli treated with protamine and exposed to an isotonic solution of Dextran 250 but not in control glomeruli. These results validate this high throughput method to measure changes in dσAlb in response to drug administration and between normal animals and those with proteinuria and renal disease.NIH grants: HL 36279 and HL 29587