Lack of ER Stress in NHERF1‐Deficient Proximal Tubule Cells Exhibiting Aberrant Npt2a Localization

Abstract

Background NHERF1 (Na+-hydrogen exchanger regulatory factor isoform 1) anchors Npt2a (Na+-dependent phosphate cotransporter type IIa) in the brush border membrane (BBM) of proximal tubules through its cross-linking of Npt2a with the actin-filament binding protein, Ezrin. In the absence of NHERF1, Npt2a accumulates aberrantly in a subapical compartment. We have demonstrated that Npt2a associates with NHERF1 in the ER/Golgi as well as the BBM, suggesting a critical role for NHERF1 in the forward trafficking of Npt2a from the ER/Golgi to the BBM. Because increased levels of ER stress have been associated with intracellular accumulation of mis-localized proteins, we hypothesized that NHERF1 deficient proximal tubule cells may experience chronic ER stress associated with mis-localization of Npt2a. Methods We measured protein expression of four ER stress markers (phosphorylated eIF2α, GRP78, GRP94, and p58IPK) in kidney cortex isolated from C57BL/6J wild type (WT) mice and their NHERF1 knock-out (KO) littermates by immunoblot and mRNA levels by RNAseq. We expressed a fluorescently labeled WT Npt2a and NHERF1 or a Npt2a/NHERF1 chimera substituting the C terminal PDZ binding motif of Npt2a with the ERM (Ezrin-Radixin-Moesin) binding domain of NHERF1 in human embryonic kidney (HEK), opossum kidney (OK), and NHERF1-deficient opossum kidney (OKH) cells. We compared WT vs chimera protein expression by confocal microscopy and protein function by radiolabeled phosphate uptake. Results We found no differences in kidney cortex protein or mRNA expression of these ER stress markers between WT and KO animals. Confocal microscopy of the Npt2a chimera expressed in HEK, OK, and OKH cells exhibited enhanced apical membrane localization when compared to WT Npt2a in all three cell types. However, no increase in radiolabeled phosphate uptake was observed between cells expressing the Npt2a chimera or wild type Npt2a. Conclusions The lack of an ER stress response suggests that despite association between NHERF1 and Npt2a in the ER/Golgi, NHERF1 is not required for normal Npt2a folding. Enhanced apical expression of the Npt2a chimera confirms the essential role of Ezrin in BBM localization of Npt2a, however, appropriate Npt2a function requires the presence of more than the ERM binding domain of NHERF1, possibly involving the association of other unidentified proteins.