Inhibition of Na⁺‐H⁺ exchange impairs receptor‐mediated albumin endocytosis in renal proximal tubule‐derived epithelial cells from opossum

Abstract

Receptor‐mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell‐surface receptor expression. In renal proximal tubules, receptor‐mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand‐receptor dissociation, vesicle trafficking, fusion events and coat formation. Here we show that the activity of Na+‐H+ exchanger isoform 3 (NHE3) is important for proper receptor‐mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+‐H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+‐H+ exchange. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. Thus, NHE3‐mediated acidification along the first part of the endocytic pathway plays an important role in receptor‐mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor‐mediated endocytosis.

Regulation of the V-ATPase in kidney epithelial cells: dual role in acid–base homeostasis and vesicle trafficking

“…While degradative pathways exist in most cell types, kidney proximal tubule cells have a particularly extensive apical endocytotic apparatus that is involved in the reabsorption and degradation of filtered proteins, and is also part of the extensive recycling of many critical apical plasma membrane proteins (Christensen, 1982;Christensen and Birn, 2002;Marshansky et al, 2002). Disruption of the V-ATPasemediated vesicle acidification process in proximal tubule cells using a variety of inhibitors, including bafilomycin, FCCP and NH 4 Cl, as well as Na + /H + exchanger 3 (NHE3) inhibition (Gekle et al, 1999), leads to diminished reabsorption of albumin (Marshansky et al, 2002) and other proteins, such as IgG light chains (Batuman and Guan, 1997) (Fig. 8).…”

Edema in the Nephrotic Syndrome

“…Perhaps the main implications are in another direction: linkage to fibrosis and inflammation in some nephrotic diseases. Previous studies have linked increased albumin endocytosis (protein overloading) in the proximal tubule and NHE3 (13); inhibition of NHE3 in vitro decreases albumin endocytosis (13), and NHE3-dependent albumin endocytosis is partially linked to activation of nuclear factor-B (NF-B) (14). Therefore, activation of NHE3 by albumin may be related to the renal inflammation and fibrosis, which has been postulated to lead to both progression of renal disease and salt retention (15,16).…”

Regulation of Expression and Localisation of the Na+/H+ Exchanger (NHE) 3 and the NHE Regulatory Factor 2 in Baboon Placental Syncytiotrophoblast by Oestrogen

“…Using immortalized kidney cell lines, others have shown that NHE3, which is abundantly expressed in and localized to endosomes in the cytoplasm and in the juxtanuclear compartment, functions at these sites in part to acidify endosomes and to facilitate vesicular fusion [22,23]. Moreover, it has been proposed that the trafficking of NHE3 to/from the cell membrane and the juxtanuclear compartment [24] represents a physiologic mechanism by which NHE3 controls Na + /H + exchange in epithelial cells of the kidney, colon, small intestine and gallbladder [25,26].…”