Kidney Injury Stimulates Apical Translocation of NHERF1

Abstract

BackgroundNHERF1 (Na+‐hydrogen exchanger regulatory factor isoform 1), recognized as a scaffold protein that regulates proximal tubule phosphate transport, also has a role in regulation of cell metabolism and survival. Our laboratory previously demonstrated more severe cisplatin‐induced acute kidney injury (AKI) in the global NHERF1 knock out (KO) mouse compared to the cisplatin‐treated wild type (WT) mouse. In the present study, we tested the hypothesis that kidney injury results in decreased NHERF1 expression by examining NHERF1 expression in two models of AKI (cisplatin and bilateral renal ischemia‐reperfusion) and two models of chronic kidney disease or CKD (adenine diet and Col4a3‐/‐) by immunofluorescent microscopy and western blot analysis.MethodsCisplatin‐induced AKI:Two‐month‐old male and female C57BL/6J WT mice received a single intraperitoneal injection of saline (vehicle) or 20 mg/kg cisplatin. Vehicle‐ and cisplatin‐treated mice were euthanized after 72 hours. Bilateral renal ischemia‐reperfusion injury AKI: Two‐month‐old male and female C57Bl/6J WT mice underwent bilateral renal artery clamp for 30 minutes followed by release. Sham and IRI surgery treated mice were euthanized at 2, 4, and 14 days post‐surgery. Adenine‐induced CKD: Two‐month‐old male and female C57Bl/6J WT mice received either regular or adenine‐supplemented chow for 20 weeks followed by euthanasia. Col4a3‐/‐ CKD: Two‐month‐old male and female C57Bl/6J heterozygous and homozygous KO mice for the collagenase type IV alpha 3 chain were followed for up to 7 months followed by euthanasia. In all four mouse models, the kidneys were removed, de‐capsulated, immersed in 3.7% formaldehyde in phosphate‐buffered saline for 24 hours, then transferred to 70% ethanol for paraffin embedding and immunofluorescent microscopy. Western blots of kidney lysates from each kidney injury model were electrophoresed in 4‐12% Bis‐Tris gradient gels and immunoblotted for NHERF1.ResultsImmunofluorescent microscopy revealed increased staining for NHERF1 at or near the apical membrane of proximal tubule epithelial cells in all four mouse injury models. For the IRI model, longitudinal immunohistochemistry demonstrated a progressive increase in the apical NHERF1 signal from baseline through day 4 post‐surgery followed by a decrease to baseline levels by day 14. Western blot analysis of lysates from all four kidney injury models showed no change in total expression of NHERF1.ConclusionsWe conclude that kidney injury results in translocation of NHERF1 to the apical membrane. These findings suggest that NHERF1 plays a significant role in the response to kidney injury, though at this time, neither the mechanism for translocation nor the specific role of NHERF1 in the response to injury is clear.