IDENTIFICATION OF ADP-RIBOSYLATION FACTOR-6 IN BRUSH-BORDER MEMBRANE AND EARLY ENDOSOMES OF HUMAN KIDNEY PROXIMAL TUBULES

The expression and distribution of ADP-ribosylation factor (ARF) small GTP-binding proteins in kidney tissue was examined, Various anti-ARF antibodies were raised against purified rec-ARF 1 and rec-ARF 6 and th eir specificity was determined, Using indirect immunofluorescence anal ysis of intact kidney, ARF proteins were found to be predominantly exp ressed in kidney tubules as compared to glomeruli. This result was fur ther supported by sodium dodecyl sulfate-polyacrylamide gel electropho resis (SDS-PAGE) and Western blot analysis of purified human kidney gl omeruli and proximal tubules. Both ARF 1 and ARF 6 were detected in pu rified human glomeruli and proximal tubules; however, ARF 1 was more a bundant than ARF 6 in these kidney structures. Brush-border membrane v esicles (BBMV) and early endosomes (EE) derived from the receptor-medi ated endocytosis pathway were isolated from purified proximal tubules of rat, dog and human kidney using a combination of magnesium precipit ation and wheat-germ agglutinin negative selection techniques. We demo nstrated that ARF 6 is associated with BBMV and with EE derived from r eceptor-mediated endocytosis pathway of human kidney proximal tubules. Using a combination of SDS-PAGE and quantitative enhanced chemilumine scence Western blot analysis, the quantification of the ARF 6 distribu tion in membrane and cytoplasmic fractions of proximal tubules was mad e and its predominance in membrane fractions was demonstrated. By anal ogy with the functional role of ARF 1 in Golgi protein transport, we s uggest that ARF 6 may play an important role in the regulation of rece ptor-mediated endocytosis and protein reabsorption by kidney proximal tubules.