The Role of Intercalated Cell Nedd4–2 in BP Regulation, Ion Transport, and Transporter Expression

Abstract

BackgroundNedd4–2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4–2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4–2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4–2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4–2 null mice by breeding global Nedd4–2 null (Nedd4–2−/−) mice with global pendrin null (Slc26a4−/−) mice. Mice ate a diet with 1%–4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl− and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro. Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4–2 gene ablation markedly increased electroneutral Cl−/HCO3− exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4–2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl−/HCO3− exchanger (Slc4a8). However, IC Nedd4–2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4–2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4–2 knockout mice.Conclusions IC Nedd4–2 regulates Cl−/HCO3− exchange in ICs., Nedd4–2 gene ablation increases BP in part through its action in these cells.