IRBIT Mediates Trafficking and Activation of Na+,K+‐ATPase by Angiotensin II

Abstract

Na+ reabsorption by the kidney is tightly regulated to maintain Na+, fluid and blood pressure homeostasis. Angiotensin II (Ang II) plays a critical role in Na+ reabsorption in the kidney during health and diseases conditions. Na+,K+‐ATPase on the basolateral membrane is one of the important targets subjected to Ang II regulation. However, the mechanism by which Ang II activates Na+,K+‐ATPase remains poorly understood. We have previously shown that IRBIT (inositol 1,4,5‐triphosphate receptor binding protein released with inositol 1,4,5‐triphosphate) binds and activates Na+/H+ exchanger (NHE3) in response to Ang II. By mass spectrometry analysis, we identified IRBIT as a novel binding protein of Na+,K+‐ATPase, prompting us hypothesize that IRBIT might coordinate transcellular Na+ absorption by regulating apical NHE3 and basolateral Na+,K+‐ATPase. Indeed, overexpression of IRBIT resulted in a greater activation of Na+,K+‐ATPase activity by Ang II in OKP cells, whereas lentiviral shRNA‐mediated knockdown of IRBIT significantly attenuated the stimulatory effect of Ang II. Confocal microscopic analysis showed colocalization of IRBIT and Na+,K+‐ATPase in the basolateral membrane and cytoplasm of OKP cells. Native‐PAGE analysis showed that IRBIT and Na+,K+‐ATPase are co‐expressed in large protein complexes of 350 and 650 kD. Importantly, IRBIT‐Na+,K+‐ATPase association was acutely enhanced by Ang II. Moreover, knockdown of IRBIT abrogated AngII‐induced membrane expression of NKA. In conclusion, IRBIT associates with Na+,K+‐ATPase and mediates Ang II‐induced membrane localization of Na+,K+‐ATPase. (This work was supported by AHA‐SDG grant)