Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton
Tetsuro Takeda, … , Robert A. Orlando, Marilyn G. Farquhar
Tetsuro Takeda, … , Robert A. Orlando, Marilyn G. Farquhar
Published July 15, 2001
Citation Information: J Clin Invest. 2001;108(2):289-301. https://doi.org/10.1172/JCI12539.
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Article

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

Abstract

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na+/H+-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside–, protamine sulfate–, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium.

Authors

Tetsuro Takeda, Tammie McQuistan, Robert A. Orlando, Marilyn G. Farquhar

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Figure 4

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Localization of NHERF1, NHERF2, PC, and ezrin in the normal rat kidney. ...
Localization of NHERF1, NHERF2, PC, and ezrin in the normal rat kidney. Strong staining for both NHERF2 (a and d) and PC (b and e) is seen in GECs in the glomerulus. PC is also present in the endothelium of peritubular capillaries (b, arrowhead) where NHERF2 is not seen. The yellow signal in the merged images (c and f) demonstrate overlap in the staining of GECs. In contrast, NHERF1 (g) is expressed mainly in the apical region of proximal tubules (arrowheads) and is not seen in glomeruli where PC is present (h and i). NHERF2 (j and m) also colocalizes with ezrin (k and n) in GECs where their staining overlaps (yellow staining in l and o). Ezrin is also expressed in proximal tubules (k, arrowheads) where NHERF1 (g) but not NHERF2 (l) is detected. Rat kidneys were processed for semithin (0.5 μm) cryosectioning, followed by double staining with mAbs anti-PC (5A) or anti-ezrin (3C12) and polyclonal anti-NHERF1 or anti-NHERF2, as described in Methods. Bars: (a–c and g–l) 20 μm; (d–f and m–o) 5 μm.