Am J Physiol Cell Physiol AJP: Heart and Circulatory Physiology
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Am J Physiol Cell Physiol 286: C768-C778, 2004. First published December 18, 2003; doi:10.1152/ajpcell.00250.2003
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

Jaafar El Annan,1 Dennis Brown,1,2 Sylvie Breton,1,2 Sylvain Bourgoin,3 Dennis A. Ausiello,1,2 and Vladimir Marshansky1,2

1Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, 2Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129-2020; 3Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Université Laval, Sainte-Foy, Quebec, Canada G1V 4G2

Submitted 16 June 2003 ; accepted in final form 10 December 2003

ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.

protein trafficking; immunofluorescence microscopy; Western blotting; endocytosis


Address for reprint requests and other correspondence: V. Marshansky, Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Harvard Medical School, 149, 13th St., CNY-8, Suite 8203, Boston, MA, 02129-2020 (E-mail: vladimir_marshansky{at}hms.harvard.edu).






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