The Role of Intercalated Cell <i>Nedd4-2</i> in BP Regulation, Ion Transport, and Transporter Expression.

Abstract

<b>Background</b><i>Nedd4-2</i> 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 <i>Nedd4-2</i> and BP. In this study, we explored the effect of intercalated cell (IC) <i>Nedd4-2</i> gene ablation on IC transporter abundance and function and on BP.<b>Methods</b> We generated IC <i>Nedd4-2</i> knockout mice using Cre-lox technology and produced global pendrin/<i>Nedd4-2</i> null mice by breeding global <i>Nedd4-2</i> null (<i>Nedd4-2^-/-</i> ) mice with global pendrin null (<i>Slc26a4^-/-</i> ) 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 <i>in vitro</i> Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.<b>Results</b> IC <i>Nedd4-2</i> 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 <i>Nedd4-2</i> gene ablation did not increase the abundance of type B IC transporters, such as AE4 (<i>Slc4a9</i>), H⁺-ATPase, barttin, or the Na⁺-dependent Cl^-/HCO₃^- exchanger (<i>Slc4a8</i>). However, IC <i>Nedd4-2</i> 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 <i>Nedd4-2</i> gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global <i>Nedd4-2</i> knockout mice.<b>Conclusions</b> IC <i>Nedd4-2</i> regulates Cl^-/HCO₃^- exchange in ICs., <i>Nedd4-2</i> gene ablation increases BP in part through its action in these cells.