Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index

Abstract

Solute reflection coefficients σ of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of σ. We determined σ NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone ( M r 40 000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a σ NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again σ NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.