Interaction between cationic dyes and erythrocyte membranes in minimal change nephropathy: an electrophoretic approach

Abstract

A study was undertaken to clarify the usefulness of two cationic dyes, alcian blue (AB) and ruthenium red (RR) in demonstrating the defect in cellular membranes noted in minimal change nephropathy (MCN). The binding of both dyes to RBC membranes purified from normal and nephrotic children was evaluated by electrophoretic titration curves. When examined separately, AB was found to precipitate spontaneously, producing macro-aggregates with no electrophoretic mobility at pH 5. This was presumed to be the result of hydrophobic interaction of the dye with itself. The same phenomenon was observed when this dye was incubated at 37°C with RBC ghost's from normal children, when AB presented a sigmoidal curve with a net positive charge for pHs higher than 5.5 and lower than 5 and no electrophoretic mobility at pH 5. However, incubation of AB with RBC ghosts from children with MCN resulted in an improvement of the solubility of the dye which then migrated with a net positive charge along the whole gradient of pH from 3.5 to 9. The presence of zwitterionic neutral detergents such as CHAPS, but not of a charged substance such as protamine sulphate, inhibited precipitation at pH 5 when incubated with membranes from normal children, supporting the hydrophobic nature of the phenomenon. When RR was used instead of AB, it was fully protonated (i.e. did not precipitate) when analysed alone, but when incubated with normal RBC ghosts, it also revealed no electrophoretic mobility at pH 5. Likewise, as seen with AB, the incubation of RR with RBC ghosts from children with MCN or with zwitterionic detergents produced an improvement in the ionization of the dye. We conclude that RBC membranes purified from children with MCN improved the solubility of AB in aqueous media by inhibiting the hydrophobic interaction of the dye, and the same explanation may hold true for the improved solubility noted with the use of RR under similar conditions. The identification of the membrane factor(s) which is responsible for this effect may be relevant to the understanding of any cellular membrane alteration in the glomeruli of children with MCN.