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Dietary NaCl is closely linked to overall blood pressure and the proximal tubule of kidney is critical in reabsorption of NaCl, bicarbonate, and water. Luminal membrane Na+/H+ exchanger, NHE3, contributes either directly or indirectly to these processes. Previous studies have documented that angiotensin II (ANGII) plays a critical role in regulation of renal NaCl reabsorption by targeting NHE3, but the underlying molecular mechanism is incompletely understood. Recently, we identified inositol 1,4,5-triphosphate receptor binding protein released with inositol1,4,5-triphosphate (IRBIT) as a novel NHE3 binding protein. We therefore investigated whether IRBIT mediates NHE3 regulation by ANGII by using opossum kidney proximal tubule cell (OKP). Our results show that knockdown of IRBIT abrogated ANGII-induced activation of NHE3, whereas overexpression of IRBIT resulted in a greater activation of NHE3 by 1 nM ANG II. In addition, the interaction between IRBIT and NHE3 was enhanced by ANGII treatment at as early as 5 min. We also determined whether Na+/H+ exchanger regulatory factor 1 (NHERF1), another NHE3 binding protein, regulates IRBIT-mediated ANGII activation of NHE3 by competing with IRBIT in binding of NHE3. The results show that overexpression of NHERF1 in OKP cells significantly decreased IRBIT-NHE3 interaction under basal conditions. In addition, NHERF1 overexpression attenuated ANGII-induced IRBIT-NHE3 interaction. Furthermore, ANGII-induced activation of NHE3 transport activity was significantly decreased when NHERF1 was overexpressed as compared to the control. In conclusion, IRBIT is an important mediator of ANGII-induced regulation of NHE3, and this effect is potentially antagonized by NHERF1 expression.
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