Original Paper

Osteopontin Traffic in Hypoxic Renal Epithelial Cells
Dierk J. Hampel^a,c, Christine Sansome^a, Victor I. Romanov^a, Aaron J. Kowalski^b, David T. Denhardt^b, Michael S. Goligorsky<sup>a,d

a</sup>Departments of Medicine and Physiology, Division of Nephrology and Hypertension, State University of New York at Stony Brook, Stony Brook, N.Y., and
^bDepartment of Cell Biology and Neuroscience, Rutgers University, Piscataway, N.J., USA;
^cCharité, Campus Virchow-Klinikum, Humboldt University at Berlin, Department of Medicine, Division of Nephrology and Medical Intensive Care, Berlin, Germany;
^dDepartment of Medicine, Nephrology Division and Renal Research Institute, New York Medical College, Valhalla, N.Y., USA

Address of Corresponding Author

Nephron Exp Nephrol 2003;94:e66-e76 (DOI: 10.1159/000071285)

  Key Words

• Osteopontin
• Hypoxia
• ATP depletion
• Renal proximal tubular cells
• Kidney

  Abstract

Osteopontin (OPN), a secretory RGD-containing phosphoprotein, is induced in acute renal injury where it plays a renoprotective role. To investigate in depth the mode of OPN secretion under stress conditions, we analyzed OPN traffic in human renal proximal tubular epithelial cells (RPTEC). Western blot analysis and fluorescence microscopy revealed trace amounts of OPN in intact cells, whereas cytoplasmic OPN levels were significantly increased after 24-48 h hypoxia. Immunoelectron microscopy of RPTEC showed predominantly apical localization of gold-labeled OPN under normal conditions. Hypoxia (24 h) increased 2.5-fold immunodetectable gold-labeled OPN at the apical plasma membrane; further reoxygenation (2 h) augmented apical and basolateral labeling 2- and 10-fold, respectively. Analysis of apical and basolateral medium conditioned by RPTEC grown on semipermeable membranes using a specially developed ELISA showed a global decrease in secreted OPN after hypoxia, which recovered following 2 h reoxygenation. Agents known to disrupt the function of the Golgi apparatus (brefeldin A, monensin) or actin cytoskeleton (cytochalasin B) significantly inhibited OPN-GFP secretion in normoxic cells. In cells recovering from hypoxia, however, OPN secretion required functional Golgi apparatus, but was not affected by cytochalasin B. These findings demonstrate that stress inhibits OPN secretion by the process dependent on the functional Golgi apparatus and actin cytoskeleton; recovery restores OPN secretion, although its polarity may become perturbed.

Copyright © 2003 S. Karger AG, Basel

  Author Contacts

Dierk J. Hampel
Charité, Campus Virchow-Klinikum, Humboldt University at Berlin
Department of Medicine, Division of Nephrology and Medical Intensive Care
Augustenburger Platz 1, DE-13353 Berlin (Germany)
E-Mail dierk.hampel@charite.de

  Article Information

Received: October 3, 2002
Accepted: April 30, 2003
Number of Print Pages : 11
Number of Figures : 6, Number of Tables : 0, Number of References : 54